BrainBike peptidomimetic enables efficient transport of proteins across brain endothelium.

Protein therapeutics cannot reach the brain in sufficient amounts because of their low permeability across the blood-brain barrier. Here we report a new family of bicyclic peptide shuttles, BrainBikes, capable of increasing transport of proteins, including antibody derivatives, in a human cell-based model of the blood-brain barrier.

Site-specific antibody masking enables conditional activation with different stimuli.

Antibody therapeutics show great potential to treat a variety of diseases. Often, the dose that can be safely administered is limited by side effects that arise from the interaction with the target outside the diseased tissue. Conditionally-active antibodies provide an additional layer of selectivity to improve safety. Distinct external stimuli or internal cues enable different control strategies and applications. However, current antibody masking strategies have low transferability across stimuli. Here we propose a versatile approach to conditionally mask antibody derivatives and its application to a single chain variable fragment (scFv) against a receptor expressed on cancer stem cells in several tumours. Our strategy relies on the site-specific conjugation of a polymer to an engineered cysteine residue through a chemically-synthesised linker that can be cleaved in response to the target stimulus. We show that the masking efficiency depends on the conjugation site and the size of the mask. An optimised mask decreases antigen binding by up to 20-fold and affinity can be fully recovered upon activation by exposure to light at 365 nm or by incubation with matrix metalloproteinases overexpressed in solid tumours. This approach opens up the possibility to rapidly engineer antibodies activatable with any internal or external stimulus.

Encoding Noncanonical Amino Acids into Phage Displayed Proteins.

Phage display facilitates the evolution of peptides and proteins for affinity selection against targets, but it is mostly limited to the chemical diversity provided by the naturally encoded amino acids. The combination of phage display with genetic code expansion allows the incorporation of noncanonical amino acids (ncAAs) into proteins expressed on the phage. In this method, we describe incorporation of one or two ncAAs in a single-chain fragment variable (scFv) antibody in response to amber or quadruplet codon. We take advantage of the pyrrolysyl-tRNA synthetase/tRNA pair to incorporate a lysine derivative and an orthogonal tyrosyl-tRNA synthetase/tRNA pair to incorporate a phenylalanine derivative. The encoding of novel chemical functionalities and building blocks in proteins displayed on phage provides the foundation for further phage display applications in fields such as imaging, protein targeting, and the production of new materials.

High-affinity peptides developed against calprotectin and their application as synthetic ligands in diagnostic assays.

Common inflammatory disorders such as ulcerative colitis and Crohn's disease are non-invasively diagnosed or monitored by the biomarker calprotectin. However, current quantitative tests for calprotectin are antibody-based and vary depending on the type of antibody and assay used. Additionally, the binding epitopes of applied antibodies are not characterized by structures and for most antibodies it is unclear if they detect calprotectin dimer, tetramer, or both. Herein, we develop calprotectin ligands based on peptides, that offer advantages such as homogenous chemical composition, heat-stability, site-directed immobilization, and chemical synthesis at high purity and at low cost. By screening a 100-billion peptide phage display library against calprotectin, we identified a high-affinity peptide (Kd = 26 ± 3 nM) that binds to a large surface region (951 Å2) as shown by X-ray structure analysis. The peptide uniquely binds the calprotectin tetramer, which enabled robust and sensitive quantification of a defined species of calprotectin by ELISA and lateral flow assays in patient samples, and thus offers an ideal affinity reagent for next-generation inflammatory disease diagnostic assays.

Chemically Enhanced Peptide and Protein Therapeutics.

Proteins and peptides are on the rise as therapeutic agents and represent a higher percentage of approved drugs each year: 24% in 2021 vs [...].

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale.

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target.

Generation of a 100-billion cyclic peptide phage display library having a high skeletal diversity.

Phage display is a powerful technique routinely used for the generation of peptide- or protein-based ligands. The success of phage display selections critically depends on the size and structural diversity of the libraries, but the generation of large libraries remains challenging. In this work, we have succeeded in developing a phage display library comprising around 100 billion different (bi)cyclic peptides and thus more structures than any previously reported cyclic peptide phage display library. Building such a high diversity was achieved by combining a recently reported library cloning technique, based on whole plasmid PCR, with a small plasmid that facilitated bacterial transformation. The library cloned is based on 273 different peptide backbones and thus has a large skeletal diversity. Panning of the peptide repertoire against the important thrombosis target coagulation factor XI enriched high-affinity peptides with long consensus sequences that can only be found if the library diversity is large.

Picomole-Scale Synthesis and Screening of Macrocyclic Compound Libraries by Acoustic Liquid Transfer.

Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets, such as protein-protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale by using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced the reaction volumes, reagent consumption, and synthesis time. As a proof-of-concept, we assembled a 2700-member target-focused macrocyclic library that we could subsequently assay in the same microtiter synthesis plates, saving the need for additional transfers and deconvolution schemes. We screened the library against the MDM2-p53 protein-protein interaction and generated micromolar and sub-micromolar inhibitors. Our approach based on acoustic liquid transfer provides a general strategy for the development of macrocycle ligands.

Development of Selective FXIa Inhibitors Based on Cyclic Peptides and Their Application for Safe Anticoagulation.

Coagulation factor XI (FXI) has emerged as a promising target for the development of safer anticoagulation drugs that limit the risk of severe and life-threatening bleeding. Herein, we report the first cyclic peptide-based FXI inhibitor that selectively and potently inhibits activated FXI (FXIa) in human and animal blood. The cyclic peptide inhibitor (Ki = 2.8 ± 0.5 nM) achieved anticoagulation effects that are comparable to that of the gold standard heparin applied at a therapeutic dose (0.3-0.7 IU/mL in plasma) but with a substantially broader estimated therapeutic range. We extended the plasma half-life of the peptide via PEGylation and demonstrated effective FXIa inhibition over extended periods in vivo. We validated the anticoagulant effects of the PEGylated inhibitor in an ex vivo hemodialysis model with human blood. Our work shows that FXI can be selectively targeted with peptides and provides a promising candidate for the development of a safe anticoagulation therapy.

Generation of a Large Peptide Phage Display Library by Self-Ligation of Whole-Plasmid PCR Product.

The success of phage display, used for developing target-specific binders based on peptides and proteins, depends on the size and diversity of the library screened, but generating large libraries of phage-encoded polypeptides remains challenging. New peptide phage display libraries developed in recent years rarely contained more than 1 billion clones, which appears to have become the upper size limit for libraries generated with reasonable effort. Here, we established a strategy based on whole-plasmid PCR and self-ligation to clone a library with more than 2 × 1010 members. The enormous library size could be obtained through amplifying the entire vector DNA by PCR, which omitted the step of vector isolation from bacterial cells, and through appending DNA coding for the peptide library via a PCR primer, which enabled efficient DNA circularization by end-ligation to facilitate the difficult step of vector-insertion of DNA fragments. Panning the peptide repertoires against a target yielded high-affinity ligands and validated the quality of the library and thus the new library cloning strategy. This simple and efficient strategy places larger libraries within reach for nonspecialist researchers to hopefully expand the possible targets of phage display applications.

Protein Chemical Synthesis Combined with Mirror-Image Phage Display Yields d-Peptide EGF Ligands that Block the EGF-EGFR Interaction.

The epidermal growth factor (EGF) pathway, being overactive in a number of cancers, is a good target for clinical therapy. Although several drugs targeting the EGF receptor (EGFR) are on the market, tumours acquire resistance very rapidly. As an alternative, small molecules and peptides targeting EGF have been developed, although with moderate success. Herein, we report the use of mirror-image phage display technology to discover protease-resistant peptides with the capacity to inhibit the EGF-EGFR interaction. After the chemical synthesis of the enantiomeric protein d-EGF, two phage-display peptide libraries were used to select binding sequences. The d versions of these peptides bound to natural EGF, as confirmed by surface acoustic waves (SAWs). High-field NMR spectroscopy showed that the best EGF binder, d-PI_4, interacts preferentially with an EGF region that partially overlaps with the receptor binding interface. Importantly, we also show that d-PI_4 efficiently disrupts the EGF-EGFR interaction. This methodology represents a straightforward approach to find new protease-resistant peptides with potential applications in cancer therapy.

Branched BBB-shuttle peptides: chemoselective modification of proteins to enhance blood-brain barrier transport.

The blood-brain barrier (BBB) hampers the delivery of therapeutic proteins into the brain. BBB-shuttle peptides have been conjugated to therapeutic payloads to increase the permeability of these molecules. However, most BBB-shuttles have several limitations, such as a lack of resistance to proteases and low effectiveness in transporting large biomolecules. We have previously reported on the THRre peptide as a protease-resistant BBB-shuttle that is able to increase the transport of fluorophores and quantum dots in vivo. In this work, we have evaluated the capacity of linear and branched THRre to increase the permeability of proteins in cellular models of the BBB. With this purpose, we have covalently attached peptides with one or two copies of the BBB-shuttle to proteins in order to develop chemically well-defined peptide-protein conjugates. While THRre does not enhance the uptake and transport of a model protein in BBB cellular models, branched THRre peptides displaying two copies of the BBB-shuttle result in a 2.6-fold increase.

From venoms to BBB-shuttles. MiniCTX3: a molecular vector derived from scorpion venom.

The present study aims to develop chlorotoxin (CTX), from Giant Yellow Israeli scorpion venom, as a new BBB-shuttle. Minimised versions of CTX were prepared to reduce its complexity while enhancing its BBB-shuttle capacity and preserving its protease-resistance. MiniCTX3, a monocyclic lactam-bridge peptidomimetic, was capable of transporting nanoparticles across endothelial cell monolayers. Our results reveal animal venoms as an outstanding source of new families of BBB-shuttles.

Phage display as a tool to discover blood-brain barrier (BBB)-shuttle peptides: panning against a human BBB cellular model.

Most potential drugs for the treatment of central nervous system disorders do not cross the blood-brain barrier (BBB). Much research effort has been devoted to the discovery of new BBB-shuttle peptides-most of which have been identified by phage display. Here we report for the first time on the use of phage display against a human BBB cellular model which mimics the characteristics of the BBB. From the panning experiment of a 12-mer library, the SGVYKVAYDWQH (SGV) peptide sequence was selected and its permeability validated in the aforementioned model. Furthermore, internalization studies suggested that SGV internalizes through a clathrin-mediated mechanism and that it increases the uptake of a cargo in endothelial cells. These results highlight the usefulness of in vitro BBB models for the discovery of BBB-shuttle peptides through phage display libraries.