Alternative Protein Scaffolds Research Articles

In vivo depletion of serum IgG by an affibody molecule binding the neonatal Fc receptor

Lowering the total level of Immunoglobulin G (IgG) in circulation is a promising general treatment option for many autoimmune diseases driven by pathogenic autoantibodies. The half-life of IgG in circulation is unusually long as a consequence of its interaction with the neonatal Fc receptor (FcRn), which protects it from lysosomal degradation by cells in contact with blood. Blocking the IgG/FcRn interaction prevents FcRn-mediated rescue, which may lead to increased catabolism and a lowering of the total IgG level. Here, we find that an engineered alternative scaffold protein, an affibody molecule, interacting specifically with FcRn, is able to block the IgG/FcRn interaction in vitro. The affibody molecule (ZFcRn) was expressed alone or as a fusion to an albumin binding domain (ABD), to extend its half-life in circulation, in both cases with retained affinity and blocking potential. Repeated i.v. injections in mice of ZFcRn and ZFcRn-ABD were found to result in an up to 40% reduction of the IgG serum-level after 5 days. Potential applications of ZFcRn as a general treatment modality for autoimmune diseases are discussed.

New Techniques for Ancient Proteins: Direct Coupling Analysis Applied on Proteins Involved in Iron Sulfur Cluster Biogenesis.

Direct coupling analysis (DCA) is a powerful statistical inference tool used to study protein evolution. It was introduced to predict protein folds and protein-protein interactions, and has also been applied to the prediction of entire interactomes. Here, we have used it to analyze three proteins of the iron-sulfur biogenesis machine, an essential metabolic pathway conserved in all organisms. We show that DCA can correctly reproduce structural features of the CyaY/frataxin family (a protein involved in the human disease Friedreich's ataxia) despite being based on the relatively small number of sequences allowed by its genomic distribution. This result gives us confidence in the method. Its application to the iron-sulfur cluster scaffold protein IscU, which has been suggested to function both as an ordered and a disordered form, allows us to distinguish evolutionary traces of the structured species, suggesting that, if present in the cell, the disordered form has not left evolutionary imprinting. We observe instead, for the first time, direct indications of how the protein can dimerize head-to-head and bind 4Fe4S clusters. Analysis of the alternative scaffold protein IscA provides strong support to a coordination of the cluster by a dimeric form rather than a tetramer, as previously suggested. Our analysis also suggests the presence in solution of a mixture of monomeric and dimeric species, and guides us to the prevalent one. Finally, we used DCA to analyze interactions between some of these proteins, and discuss the potentials and limitations of the method.

Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold.

Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 μM to 1.6 μM, according to surface plasmon resonance data. While L4 has 1–2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1–0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.

64Cu-Labeled Gp2 Domain for PET Imaging of Epidermal Growth Factor Receptor.

This purpose of this study is to determine the efficacy of a 45-amino acid Gp2 domain, engineered to bind to epidermal growth factor receptor (EGFR), as a positron emission tomography (PET) probe of EGFR in a xenograft mouse model. The EGFR-targeted Gp2 (Gp2-EGFR) and a nonbinding control were site-specifically labeled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Binding affinity was tested toward human EGFR and mouse EGFR. Biological activity on downstream EGFR signaling was examined in cell culture. DOTA-Gp2 molecules were labeled with 64Cu and intravenously injected (0.6-2.3 MBq) into mice bearing EGFRhigh (n = 7) and EGFRlow (n = 4) xenografted tumors. PET/computed tomography (CT) images were acquired at 45 min, 2 h, and 24 h. Dynamic PET (25 min) was also acquired. Tomography results were verified with gamma counting of resected tissues. Two-tailed t tests with unequal variances provided statistical comparison. DOTA-Gp2-EGFR bound strongly to human (KD = 7 ± 5 nM) and murine (KD = 29 ± 6 nM) EGFR, and nontargeted Gp2 had no detectable binding. Gp2-EGFR did not agonize EGFR nor antagonize EGF-EGFR. 64Cu-Gp2-EGFR tracer effectively localized to EGFRhigh tumors at 45 min (3.2 ± 0.5%ID/g). High specificity was observed with significantly lower uptake in EGFRlow tumors (0.9 ± 0.3%ID/g, p < 0.001), high tumor-to-background ratios (11 ± 6 tumor/muscle, p < 0.001). Nontargeted Gp2 tracer had low uptake in EGFRhigh tumors (0.5 ± 0.3%ID/g, p < 0.001). Similar data was observed at 2 h, and tumor signal was retained at 24 h (2.9 ± 0.3%ID/g). An engineered Gp2 PET imaging probe exhibited low background and target-specific EGFRhigh tumor uptake at 45 min, with tumor signal retained at 24 h postinjection, and compared favorably with published EGFR PET probes for alternative protein scaffolds. These beneficial in vivo characteristics, combined with thermal stability, efficient evolution, and small size of the Gp2 domain validate its use as a future class of molecular imaging agents.

Intracellular targeting with engineered proteins.

If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action.

Abstract LB-062: Evaluation of drug conjugation sites in an anti-EGFR Centyrin

Abstract Centyrins are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with increased tissue penetration and simplified manufacturing. To identify optimal positions for chemical conjugation within the Centyrin scaffold, the crystal structure of an anti-EGFR Centyrin was solved and a library consisting of all possible single-cysteine variants was constructed and assessed for expression level in E. coli, efficiency of conjugation to maleimide derivatives, protein aggregation, thermostability, and EGFR binding. The best variants were conjugated to cytotoxic drugs and compared in a cell-killing assay. Three optimal positions were identified at which the Centyrin conjugated efficiently to maleimides; retained high expression, antigen binding, and biophysical properties; and showed increased cell-killing potency relative to the parent molecule. Citation Format: Rosa Cardoso, Shalom Goldberg, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven Jacobs, Gary Gilliland, Karyn O’Neil. Evaluation of drug conjugation sites in an anti-EGFR Centyrin. [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 LB-062.

Chemical shift assignment of the alternative scaffold protein IscA.

The IscA protein (11.5 kDa) is an essential component of the iron sulphur cluster biogenesis machine. In bacteria, the machine components are clustered in operons, amongst which the most important is the isc operon. Bacterial IscA has direct homologues also in eukaryotes. Like the protein IscU, IscA is thought to assist cluster formation as an alternative scaffold protein which receives the cluster before transferring it further to the final acceptors. Several crystal structures have been published. They all report an IscA dimeric form, although the packing of the protomers in the dimers differs amongst structures. No solution studies have currently been reported. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of the cluster-free E. coli IscA as a starting point for further studies of the structure and functions of this still poorly characterized protein. We show that IscA exists in solution as an equilibrium between different species. Spectrum assignment was thus challenging given the heterogeneous nature of the sample but doable through judicious choice of selective labelling and concentration dependent studies.

Library of binding protein scaffolds (LibBP): a computational platform for selection of binding protein scaffolds.

Developments in biotechnology have enabled the in vitro evolution of binding proteins. The emerging limitations of antibodies in binding protein engineering have led to suggestions for other proteins as alternative binding protein scaffolds. Most of these proteins were selected based on human intuition rather than systematic analysis of the available data. To improve this strategy, we developed a computational framework for finding desirable binding protein scaffolds by utilizing protein structure and sequence information. For each protein, its structure and the sequences of evolutionarily-related proteins were analyzed, and spatially contiguous regions composed of highly variable residues were identified. A large number of proteins have these regions, but leucine rich repeats (LRRs), histidine kinase domains and immunoglobulin domains are predominant among them. The candidates suggested as new binding protein scaffolds include histidine kinase, LRR, titin and pentapeptide repeat protein. The database and web-service are accessible via http://bcbl.kaist.ac.kr/LibBP CONTACT: kds@kaist.ac.krSupplementary data: Supplementary data are available at Bioinformatics online.

Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein’s scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

Artificial affinity proteins as ligands of immunoglobulins.

A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized.

Abstract 3222: Structural evaluation of several antitumor Tencon variants

Abstract Tencons are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with the biophysical features of smaller molecules and hold high promise as biotherapeutic agents. We present the crystal structures and antigen binding data of several anti-Tumor Tencon variants. The structures of the variants show a switch of the FG loop conformation from α-helical at the tip to mostly β-strands. This conformational change prompts differential exposure of potential binding residues and a finger-like shape for the FG region that may facilitate its access to binding pockets on the target protein, thus likely accounting for the observed differences in KD. Citation Format: Rosa Cardoso, Shalom Goldberg, Jinquan Luo, Alexander Barnakov, Edward Swift, Steven Jacobs, Gary Gilliland. Structural evaluation of several antitumor Tencon variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3222. doi:10.1158/1538-7445.AM2014-3222

Fluorescent fusion proteins derived from the tenth human fibronectin domain

Hybrid molecules of a new type bearing a red fluorescent protein mCherry and one of the alternative scaffold proteins, the 10th human fibronectin type III domain (10Fn3), which can be used for the construction of antibody mimics with various binding specificity, were obtained. Different variants of the gene encoding the hybrid fluorescent protein were constructed and their expression in Escherichia coli cells was studied. The mCherry N-terminal position and the modification of its N-terminal amino acid sequence proposed were shown to promote the efficient bacterial expression of the hybrid protein in the soluble form. On the basis of the proposed construct a hybrid fluorescent protein ChIBF containing an αVβ3-integrin binding variant of 10Fn3 was obtained, and its use for the visualization of αVβ3-integrin at the surface of MDCK epithelial cells was demonstrated by confocal microscopy.

Alternative scaffold proteins

Review is devoted to the challenging direction in modem molecular biology and bioengineering - the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP molecules incorporate conservative protein core and hypervariable regions, providing for the binding function. Structural classification of ASP includes several types which differ also in their molecular targets and potential applications. Construction of artificial binding proteins on the ASP basis implies a combinatorial library design with subsequent selection of specific binders with the use of phage display or the modem cell-free systems. Alternative binding proteins on non-immunoglobulin scaffolds find broad applications in different fields ofbiotechnology and molecular medicine.

Abstract 3632: Nonclinical pharmacokinetics and safety of Angiocal, the first VEGF-A-specific alternative scaffold protein entering clinical development for cancer

Abstract Anticalins are high affinity scaffold proteins based on human lipocalins and represent a new class of biologics. Although lipocalins share limited sequence identity among each other they are all made up of a single conserved beta-barrel. The four loops protruding from the opening of the calyx contribute to the observed various binding functions of different lipocalins. The loop residues of human tear lipocalin were randomized to generate a combinatorial library and successive rounds of panning and screening were employed to generate an Anticalin with high affinity and specificity for VEGF-A, Angiocal. Angiocal was found to antagonize VEGF-A function by preventing its interaction with cognate membrane receptors. Following the initial characterization in cell-based and in vivo models as communicated previously (AACR 2008 #4077, AACR 2009 #2318) we sought to estimate the human pharmacokinetics of Angiocal by interspecies scaling. Therefore, PK profiles in mice, rats and cynomolgus monkeys were obtained and will be presented. Therapeutic antibodies such as bevacizumab are usually well tolerated while significant toxicities occur in a subset of patients, for example leading to an enhanced risk for thromboembolic events. It has been demonstrated that bevacizumab forms multimeric immune complexes in vivo which may be a cause for these effects. Furthermore, immune complex deposition in glomeruli of the kidney may cause glomerulosclerosis. With respect to thromboembolic complications of bevacizumab use, these were mirrored in human FcgammaIIa receptor transgenic mice where administration of complexes between heparin, Bevacizumab and heparin-binding isoforms of its VEGF-A target lead to platelet aggregation and thrombosis (Meyer, T. et al. J. Thromb. Haemost. 2009, 7: 171-181). Angiocal was tested in the same model and does not cause any of these adverse effects on platelets in vivo. GLP toxicology studies with Angiocal were performed in two relevant species to determine the NOAEL. In addition, we did not observe effects on inflammatory cytokines or complement factors in these studies while exaggerated pharmacodynamic effects consistent with potent VEGF neutralization were seen. Angiocal is currently undergoing phase Ib clinical testing in cancer patients to establish Anticalins as novel and safe class of therapeutic molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2011-3632

Kinesins at a glance.

Kinesin was discovered in 1985 – 25 years ago – based on its motility in cytoplasm extruded from the giant axon of the squid ([Allen et al., 1982][1]; [Brady et al., 1982][2]; [Vale et al., 1985][3]). The purified protein, now known as kinesin-1, consisted of two heavy chains with motor activity

Directed Evolution of ATP Binding Proteins from a Zinc Finger Domain by Using mRNA Display

Antibodies have traditionally been used for isolating affinity reagents to new molecular targets, but alternative protein scaffolds are increasingly being used for the directed evolution of proteins with novel molecular recognition properties. We have designed a combinatorial library based on the DNA binding domain of the human retinoid-X-receptor (hRXRalpha). We chose this domain because of its small size, stable fold, and two closely juxtaposed recognition loops. We replaced the two loops with segments of random amino acids, and used mRNA display to isolate variants that specifically recognize adenosine triphosphate (ATP), demonstrating a significant alteration of the function of this protein domain from DNA binding to ATP recognition. Many novel independent sequences were recovered with moderate affinity and high specificity for ATP, validating this scaffold for the generation of functional molecules.

Beyond antibodies ? lessons from bacterial ?immunity?

Isolation and production of highly specific protein-based binding molecules are crucial to the ever expanding diagnostics, therapeutics and protein array fields. Traditionally, such reagents have been sourced from vertebrate immune systems, where antibodies have evolved over millennia into highly effective molecules of immune surveillance capable of targeting a huge range of targets in response to infection and disease. Now, a growing number of alternative protein scaffolds are being investigated as specific binding molecules incorporating a diverse and powerful range of binding and recognition interfaces. These are being sourced from human proteins, from alternative immune molecules present in evolutionarily old vertebrates, and from highly evolved binding proteins in prokaryotic systems.

Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways.

tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s(2)C), 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms(2)io(6)A (10-fold) and s(2)C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms(2)io(6)A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s(4)U and the s(2)U moiety of (c)mnm(5)s(2)U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s(2)C and MiaB in the synthesis of ms(2)io(6)A) in the transfer of sulfur to the tRNA.

Subcellular compartmentalization of human Nfu, an iron-sulfur cluster scaffold protein, and its ability to assemble a [4Fe-4S] cluster.

Iron-sulfur (Fe-S) clusters serve as cofactors in many proteins that have important redox, catalytic, and regulatory functions. In bacteria, biogenesis of Fe-S clusters is mediated by multiple gene products encoded by the isc and nif operons. In particular, genetic and biochemical studies suggest that IscU, Nfu, and IscA function as scaffold proteins for assembly and delivery of rudimentary Fe-S clusters to target proteins. Here we report the characterization of human Nfu. A combination of biochemical and spectroscopic techniques, including UV-visible absorption and 57Fe Mössbauer spectroscopies, have been used to investigate the ability of purified human Nfu to assemble Fe-S clusters. The results suggest that Nfu can assemble approximately one labile [4Fe-4S] cluster per two Nfu monomers, and support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. Analyses of genomic DNA, transcripts, and translation products indicate that alternative splicing of a common pre-mRNA results in synthesis of two Nfu isoforms with distinct subcellular localizations. Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. These results, together with previous reports of subcellular distributions of isoforms of human IscS and IscU in mitochondria, cytosol, and nucleus suggest that the Fe-S cluster assembly machineries are compartmentalized in higher eukaryotes.

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding.

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology.