Engineered Protein Scaffolds Research Articles

A Bioengineered Stable Protein 1‐Hemin Complex with Enhanced Peroxidase‐Like Catalytic Properties

Enzymes have gained their unique efficiency and catalytic activity through billions of years of evolution, perfecting their active site to a desired reaction. Inspired by nature, a novel enzyme‐mimicking platform is designed based on stable protein 1 (SP1) to create a nano‐compartment that mimics peroxidase activity. The biohybrid reveals enhanced activity over the hemin cofactor alone and improved stability in organic solvents in comparison to native peroxidase. Furthermore, the utilization of the obtained biohybrid in an optical glucose biosensing platform is shown. The biohybrid crystallographic structure is solved, indicating that the SP1 structure is not affected by the hemin coordination. This work opens the path for developing new cofactor binding centers in engineered protein scaffolds for various artificial catalytic processes.

Abstract 1688: Characterization of severe glaucoma-associated myocilin mutations in an engineered protein scaffold

Abstract 1688: Characterization of severe glaucoma-associated myocilin mutations in an engineered protein scaffold

Ferritin nanocages: a versatile platform for nanozyme design.

Nanozymes are a class of nanomaterials with enzyme-like activities and have attracted increasing attention due to their potential applications in biomedicine. However, nanozyme design incorporating the desired properties remains challenging. Natural or genetically engineered protein scaffolds, such as ferritin nanocages, have emerged as a promising platform for nanozyme design due to their unique protein structure, natural biomineralization capacity, self-assembly properties, and high biocompatibility. In this review, we highlight the intrinsic properties of ferritin nanocages, especially for nanozyme design. We also discuss the advantages of genetically engineered ferritin in the versatile design of nanozymes over natural ferritin. Additionally, we summarize the bioapplications of ferritin-based nanozymes based on their enzyme-mimicking activities. In this perspective, we mainly provide potential insights into the utilization of ferritin nanocages for nanozyme design.

Recent Advances in the Scaffold Engineering of Protein Binders.

In recent years, extensive attention has been given to the generation of new classes of ligand- specific binding proteins to supplement monoclonal antibodies. A combination of protein engineering and display technologies has been used to manipulate non-human antibodies for humanization and stabilization purposes or even the generation of new binding proteins. Engineered protein scaffolds can now be directed against therapeutic targets to treat cancer and immunological disorders. Although very few of these scaffolds have successfully passed clinical trials, their remarkable properties such as robust folding, high solubility, and small size motivate their employment as a tool for biology and applied science studies. Here, we have focused on the generation of new non-Ig binding proteins and single domain antibody manipulation, with a glimpse of their applications.

White-emitting Protein-Metal Nanocluster Phosphors for Highly Performing Biohybrid Light-Emitting Diodes.

This work presents a simple in situ synthesis and stabilization of fluorescent gold nanoclusters (AuNCs) with different sizes using engineered protein scaffolds in water. The protein-AuNC hybrids show a dual emission (450 and 700 nm) with a record photoluminescence quantum yield of 20%. These features impelled us to apply them to biohybrid light-emitting diodes as color down-converting filters or biophosphors. Efficient white emission (x/y CIE color coordinates of 0.31/0.29) and stabilities of more than 800 h were achieved. This represents a 2 orders of magnitude enhancement compared to the prior art. Besides the outstanding performance, the protein scaffold also infers a unique anisotropic emission character that is considered as a proof-of-concept of high interest for single-point lighting and display.

Engineered Protein Scaffolds as Next-Generation Therapeutics.

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.

Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins.

Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5°C, pH0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.

Studies of the oligomerisation mechanism of a cystatin-based engineered protein scaffold

Engineered protein scaffolds are an alternative to monoclonal antibodies in research and drug design due to their small size, ease of production, versatility, and specificity for chosen targets. One key consideration when engineering such proteins is retaining the original scaffold structure and stability upon insertion of target-binding loops. SQT is a stefin A derived scaffold protein that was used as a model to study possible problems associated with solution behaviour of such aptamers. We used an SQT variant with AU1 and Myc insertion peptides (SQT-1C) to study the effect of peptide insertions on protein structure and oligomerisation. The X-ray structure of monomeric SQT-1C revealed a cystatin-like fold. Furthermore, we show that SQT-1C readily forms dimers and tetramers in solution. NMR revealed that these oligomers are symmetrical, with inserted loops comprising the interaction interface. Two possible mechanisms of oligomerisation are compared using molecular dynamics simulations, with domain swap oligomerisation being thermodynamically favoured. We show that retained secondary structure upon peptide insertion is not indicative of unaltered 3D structure and solution behaviour. Therefore, additional methods should be employed to comprehensively assess the consequences of peptide insertions in all aptamers, particularly as uncharacterized oligomerisation may alter binding epitope presentation and affect functional efficiency.

Synthetic Control of Tertiary Helical Structures in Short Peptides.

Helical secondary and tertiary motifs are commonly observed as binding epitopes in natural and engineered protein scaffolds. While several strategies have been described to constrain α-helices or reproduce their binding attributes in synthetic mimics, general strategies to mimic tertiary helical motifs remain in their infancy. We recently described a synthetic strategy to develop helical dimers ( J. Am. Chem. Soc. 2015, 137, 11618-11621). We found that replacement of an interhelical salt bridge with a covalent bond can stabilize antiparallel motifs in short sequences. Here we show that the approach can be generalized to obtain antiparallel and parallel dimers as well as trimer motifs. Helical stabilization requires judiciously designed cross-linkers as well as optimal interhelical hydrophobic packing. We anticipate that these mimics would afford new classes of modulators of biological function.

Recognition of Oxidized 5-Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds.

Methylation of genomic cytosine to 5-methylcytosine is a central regulatory element of mammalian gene expression with important roles in development and disease. 5-methylcytosine can be actively reversed to cytosine via oxidation to 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine by ten-eleven-translocation dioxygenases and subsequent base excision repair or replication-dependent dilution. Moreover, the oxidized 5-methylcytosine derivatives are potential epigenetic marks with unique biological roles. Key to a better understanding of these roles are insights into the interactions of the nucleobases with DNA-binding protein scaffolds: Natural scaffolds involved in transcription, 5-methylcytosine-reading and -editing as well as general chromatin organization can be selectively recruited or repulsed by oxidized 5-methylcytosines, forming the basis of their biological functions. Moreover, designer protein scaffolds engineered for the selective recognition of oxidized 5-methylcytosines are valuable tools to analyze their genomic levels and distribution. Here, we review recent structural and functional insights into the molecular recognition of oxidized 5-methylcytosine derivatives in DNA by selected protein scaffolds.

Targeting Insulin Receptor in Breast Cancer Using Small Engineered Protein Scaffolds.

Insulin receptor (InsR) and the type I insulin-like growth factor (IGF1R) are homologous receptors necessary for signal transduction by their cognate ligands insulin, IGF-I and IGF-II. IGF1R mAbs, intended to inhibit malignant phenotypic signaling, failed to show benefit in patients with endocrine-resistant tumors in phase III clinical trials. Our previous work showed that in tamoxifen-resistant cells, IGF1R expression was lacking, but InsR inhibition effectively blocked growth. In endocrine-sensitive breast cancer cells, insulin was not growth stimulatory, likely due to the presence of hybrid InsR/IGF1R, which has high affinity for IGF-I, but not insulin. Combination inhibition of InsR and IGF1R showed complete suppression of the system in endocrine-sensitive breast cancer cells. To develop InsR-binding agents, we employed a small protein scaffold, T7 phage gene 2 protein (Gp2) with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5, and #10) with low nanomolar affinity and specific binding to cell surface InsR. These Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and resistant breast cancer, but did not downregulate InsR expression. Gp2 #5 and Gp2 #10 disrupted InsR function by inhibiting ligand-induced receptor activation. In contrast, Gp2 #1 did not block InsR phosphorylation. Notably, Gp2 #1 binding was enhanced by pretreatment of cells with insulin, suggesting a unique receptor-ligand-binding mode. These Gp2 variants are the first nonimmunoglobulin protein scaffolds to target insulin receptor and present compelling opportunity for modulation of InsR signaling. Mol Cancer Ther; 16(7); 1324-34. ©2017 AACR.

Practical Immuno-PET Radiotracer Design Considerations for Human Immune Checkpoint Imaging.

Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. Here, we sought to optimize noninvasive immuno-PET imaging of human programmed death-ligand 1 (PD-L1) expression, in a preclinical model, using a small high-affinity engineered protein scaffold (HAC-PD1). Six HAC-PD1 radiotracer variants were developed and used in preclinical imaging and biodistribution studies to assess their ability to detect human PD-L1 expression in vivo. Radiotracer design modifications included chelate, glycosylation, and radiometal. HACA-PD1 was adopted as the naming convention for aglycosylated tracer variants. NOD scid γ-(NSG) mice were inoculated with subcutaneous tumors engineered to either be constitutively positive (CT26 hPD-L1) or be negative (ΔmPD-L1 CT26) for human PD-L1 expression. When the tumors had grown to an average size of 1 cm in diameter, mice were injected with 0.75-2.25 MBq (∼10 μg) of an engineered radiotracer variant and imaged. At 1 h after injection, organs were harvested for biodistribution. Of the practical immuno-PET tracer modifications considered, glycosylation was the most prominent design factor affecting tracer uptake, specificity, and clearance. In imaging studies, aglycosylated 64Cu-NOTA-HACA-PD1 most accurately visualized human PD-L1 expression in vivo. We reasoned that because of the scaffold's small size (14 kDa), its pharmacokinetics may be suitable for labeling with the short-lived and widely clinically available radiometal 68Ga. At 1 h after injection, 68Ga-NOTA-HACA-PD1 and 68Ga-DOTA-HACA-PD1 exhibited promising target-to-background ratios in ex vivo biodistribution studies (12.3 and 15.2 tumor-to-muscle ratios, respectively). Notably, all HAC-PD1 radiotracer variants enabled much earlier detection of human PD-L1 expression (1 h after injection) than previously reported radiolabeled antibodies (>24 h after injection). This work provides a template for assessing immuno-PET tracer design parameters and supports the translation of small engineered protein radiotracers for imaging human immune checkpoints.

Novel electrospun nanofibers of modified gelatin-tyrosine in cartilage tissue engineering

In natural cartilage tissues, chondrocytes are linked to extracellular matrix (ECM) through cell-surface binding proteins. Surface modification of gelatin can provide a new generation of biopolymers and fibrous scaffolds with chemical, mechanical, and biological properties. In this study tyrosine protein and 1,2,3-triazole ring were utilized to functionalize gelatin without Cu catalyst. Their molecular structure was characterized by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1HNMR). Chemical cross-linkers such as glutaraldehyde (GA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) were used to electrospin the modified gelatin. The modification of gelatin and cross-linking effects were confirmed by scanning electron microscopy (SEM), contact angle measurement, and mechanical tests. MTT assay using chondrocyte cells showed cell viability of electrospun modified gelatin scaffolds. In vitro cell culture studies showed that electrospun engineered protein scaffolds would support the attachment and growth of cells. The results also showed that cross-linked nanofibers with EDC/NHS could be considered excellent matrices in cell adhesion and proliferation before electrospinning process and their potential substrate in tissue engineering applications, especially in the field of cartilage engineering.

Abstract 1881: Insulin receptor targeting in breast cancer through yeast surface display

Abstract Selective estrogen receptor modulators (SERMs) such as tamoxifen are used to treat estrogen receptor (ER) positive breast cancers, the most common intrinsic subtype. The development of secondary resistance to SERMs is an unsolved clinical dilemma, leading to the exploration, and approval, of therapies targeting growth factor signaling pathways. The insulin-like growth factor (IGF) system is well documented to cooperate with ER and is implicated as a contributor to endocrine resistance. Unfortunately, antibodies directed against Type I IGF receptor (IGF1R) failed to demonstrate clinical efficacy in endocrine resistant breast cancer. We developed a tamoxifen resistant (TamR) model to further identify the role for this system in endocrine resistance. In these cells, IGF1R levels are low but the level of insulin receptor (InsR), a closely related receptor of IGF1R still remains functional. Thus, we hypothesize that InsR may serve as a compensatory pathway to the loss of IGF1R in TamR cells and InsR may be a target in the therapy of endocrine resistant breast cancer. Indeed, our results showed that TamR breast cancer cells are more sensitive to insulin stimulation than wild-type cells. As compared to the parental cells, insulin-stimulated TamR cells showed enhanced PI3K/AKT and MAPK/ERK activation, greater monolayer and anchorage-independent growth. Suppression of InsR expression with either lentiviral shRNA or pharmacological methods in TamR cells was able to attenuate their sensitivity towards insulin-regulated PI3K/MAPK activation and growth, suggesting InsR targeting may be necessary. To target InsR, we are developing InsR-selective small protein scaffolds - the 10th type III domain of human fibronectin (Fn3) and T7 phage gene 2 protein (Gp2) using yeast surface display. This technique has arisen as an alternative candidate to bind cells surface proteins. Compared to antibodies, protein scaffolds are smaller (<12kDa), thermally more stable, lack disulfide bonds, tolerance to mutations and have the ability to bind a large variety of proteins. Using error-prone polymerase mutagenesis, we have identified improved Fn3 and Gp2 binders with increased affinity, specificity, and stability for InsR. Yeast surface displayed (YSD) Gp2 and Fn3 libraries yield affinities for recombinant InsR in the high nanomolar range and some specificity with an ability to differentiate binding from IGF1R. Purified Gp2 binder was able to distinguish InsR expression levels between InsRlow and InsRhigh mammalian cells, verifying its specificity for cell surface InsR. Its affinity for cell surface InsR, however was much lower compared to YSD affinity. Preliminary analyses also indicated that Gp2 binders have minimal effect in InsR-regulated signaling and short-term biological functions in vitro. These InsR binding engineered protein scaffolds will further be improved and evaluated as a potential imaging, diagnostic, and therapeutic tools. Citation Format: Jie Ying Chan, Kelly LaPara, Benjamin Hackel, Douglas Yee. Insulin receptor targeting in breast cancer through yeast surface display. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1881.

ANTICALIgN: visualizing, editing and analyzing combined nucleotide and amino acid sequence alignments for combinatorial protein engineering.

ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.

Rational Design of a Photoactivatable Cofilin Analog using a Novel Lov-Binding Protein

The spatial and temporal organization of protein activity is an essential but poorly understood aspect of cellular signaling. Optogenetics is emerging as a powerful tool for precise modulation of specific protein's activity within living cells and animals. For non-channel proteins, optical control of protein activity has been accomplished through light-regulated protein dimerization, oligomerization, and by light-controlled steric block of a protein or peptide active site using the LOV (light-oxygen-voltage) domain. Illumination triggers a conformational change in LOV that reversibly releases the steric block. A challenge in this approach has been the difficulty of controlling the orientation of LOV in the dark to efficiently block the protein site. Recently our lab has generated a novel LOV-binding protein based on z-affibodies, a class of engineered protein scaffold. Our z-affibody (termed Zdk) binds selectively to the dark state of LOV ( 4 μM affinity in the light). We show that Zdk can be used to control the orientation of LOV in the dark state and efficiently block the actin-binding site of cofilin in a light-dependent, reversible manner. To make photoactivatable cofilin (PA-cofilin), Zdk was fused to the N-terminus of cofilin and LOV was fused to the C-terminus. We used Rosetta-based molecular modeling to rationally design linkers that oriented the Zdk-LOV complex over the actin-binding interface and also to rationally design Zdk variants with a range of affinities for LOV. Using a Zdk of the optimum affinity for LOV was found to be critical in the design. PA-cofilin undergoes a five-fold increase in actin-binding in response to light, demonstrating the tight control of protein activity that can be achieved using Zdk. Ongoing studies using PA-cofilin to probe the role of cofilin in polarized migration will be described.

‘Engineered protein scaffolds: have they lived up to expectations?’

Monoclonal antibodies are currently the most successful class of therapeutic agents. However, the conventional immunoglobulin (Ig) format is not always optimally suited to meet clinical demands. First, immunological effector functions mediated by the Fc region can evoke undesired side effects. Second, poor tissue penetration due to the large molecular size hampers successful treatment of solid tumors. Also, the long circulation in blood resulting from both the large size and FcRn-mediated endosomal recycling is unfavorable both for therapies that require flexible adjustment of dosing and for in vivo imaging applications. Finally, due to their complex biomolecular architecture, including four polypeptide chains with around 1500 amino acids and at least two glycosylation sites, the production of full size antibodies is costly and requires mammalian expression systems. As a consequence, during the last two decades more than 50 alternative types of binding proteins have been proposed with the intention to overcome some of the inherent limitations of antibodies. However, only a minority of these ‘alternative scaffolds’ have reached the clinic so far, which can be seen as the ultimate success in pharmaceutical biotechnology. According to recent reviews, ten drug candidates based on seven different protein scaffolds have been tested in clinical trials while one biological received market approval (the engineered Kunitz domain/protease inhibitor ecallantide) [1,2]. At present, the biopharmaceutical development is dominated by the following protein scaffolds: • Affibodies based on the Z-domain of Staphylococcal protein A [3];

Rekombinante therapeutische Proteine — eine Erfolgsgeschichte

After a first decade of using recombinant versions of natural proteins, novel engineered biologics have complemented the arsenal of medicine during the last 20 years. Today, tailor-made hormones, enzymes and fusion proteins offer optimised pharmacokinetic properties and new modes of action. Beside human(ized) antibodies, engineered protein scaffolds have recently entered the pharmaceutical R&D pipeline and open prospects for the specific targeting of diverse disease targets especially in the areas of oncology and inflammation.

Potent and regularizable crosslinking of ultrafine fibrous protein scaffolds for tissue engineering using a cytocompatible disaccharide derivative.

Sucrose, a naturally-occurring disaccharide, could be oxidized to polar polyaldehydes to improve the performance properties of tissue engineering scaffolds composed of three-dimensionally arranged ultrafine protein fibers in a controllable manner. With significantly better water stability, an in vitro study demonstrated that the biocompatibility of the oxidized sucrose crosslinked scaffolds was similar to the citric acid crosslinked ones. Due to their structural similarity to the major component in native extracellular matrices (ECMs), proteins had advantages over other macromolecules for development of tissue engineering scaffolds. We have successfully developed three-dimensional (3D) ultrafine fibrous structures from proteins that could mimic the authentic 3D architectures of native ECMs. However, the enlarged contacting area exposed to water worsened the poor water stability of proteins, and thus necessitated potent and non-toxic crosslinking. Citric acid, a biobased crosslinker, was widely recognized as safe and showed good crosslinking efficiency among non-toxic crosslinkers for proteins, though was still less potent than aldehydes. In this research, sucrose was oxidized to non-volatile polyaldehydes with high polarity and low toxicity. Compared to those crosslinked with citric acid, the 3D ultrafine fibrous zein scaffolds crosslinked with oxidized sucrose showed significantly better water stability and similar cytocompatibility via an in vitro study with preosteoblasts. In summary, oxidized sucrose could be a safe and potent crosslinker to improve water stability of macromolecule-based materials for medical and industrial applications.

Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.