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.
View Article and Find Full Text PDFAntibody 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.
View Article and Find Full Text PDFPhage 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.
View Article and Find Full Text PDFProteins and peptides are on the rise as therapeutic agents and represent a higher percentage of approved drugs each year: 24% in 2021 vs [...
View Article and Find Full Text PDFPeptides show high promise in the targeting and intracellular delivery of next-generation bio- and nano-therapeutics. However, the proteolytic susceptibility of peptides is one of the major limitations of their activity in biological environments. Numerous strategies have been devised to chemically enhance the resistance of peptides to proteolysis, ranging from - and -termini protection to cyclization, and including backbone modification, incorporation of amino acids with non-canonical side chains and conjugation.
View Article and Find Full Text PDFThe high selectivity and affinity of antibody binding have made antibodies all-pervasive tools in therapy, diagnosis, and basic science. A plethora of chemogenetic approaches has been devised to make antibodies responsive to stimuli ranging from light to enzymatic activity, temperature, pH, ions, and effector molecules. Within a single decade, the field of activatable antibodies has yielded marketed therapeutics capable of engaging antigens that could not be targeted with traditional antibodies, as well as new tools to control intracellular protein location and investigate biological processes.
View Article and Find Full Text PDFProtein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity.
View Article and Find Full Text PDFBackground: Human breast milk (BM) fortification is required to feed preterm newborns with less than 32 weeks of gestation. However, addition of fortifiers increases osmolarity and osmolarity values higher than 450 mOsm/kg may be related to gastrointestinal pathology. Hence, fortifier selection and dosage are key to achieve optimal feeding.
View Article and Find Full Text PDFPhage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non-canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl-tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene-containing ncAA (CypK) at diverse sites on a displayed single-chain antibody variable fragment (ScFv), in response to amber and quadruplet codons.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFAntibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics.
View Article and Find Full Text PDFHomogeneous antibody-drug conjugates (ADCs), generated by site-specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site-specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic-code expansion.
View Article and Find Full Text PDFMost 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.
View Article and Find Full Text PDFBrain delivery is one of the major challenges in drug development because of the high number of patients suffering from neural diseases and the low efficiency of the treatments available. Although the blood-brain barrier (BBB) prevents most drugs from reaching their targets, molecular vectors - known as BBB shuttles - offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins.
View Article and Find Full Text PDFDrug delivery across the blood-brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non-invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback.
View Article and Find Full Text PDFThe blood-brain barrier (BBB) is a formidable physical and enzymatic barrier that tightly controls the passage of molecules from the blood to the brain. In fact, less than 2 % of all potential neurotherapeutics are able to cross it. Here, by applying the retro-enantio approach to a peptide that targets the transferrin receptor, a full protease-resistant peptide with the capacity to act as a BBB shuttle was obtained and thus enabled the transport of a variety of cargos into the central nervous system.
View Article and Find Full Text PDFVenoms are currently the focus of many drug discovery programs because they contain highly bioactive and selective components. Among them, apamin, a peptide found in bee venom, has received considerable attention because of its affinity for certain potassium channels and also because of its interesting structure and high stability to extreme pH and temperatures. Although apamin has long been claimed to cross the blood-brain barrier (BBB), only a few studies have been performed producing controversial results.
View Article and Find Full Text PDFLess than 2% of all potential neurotherapeutics cross the blood-brain barrier (BBB). Here, we sought to build a construct with the capacity to cross this barrier, to behave as a chemical delivery system, and, once inside the central nervous system, to be transformed and then act as an enzyme inhibitor. With all this in mind, here, we describe the entire process to obtain such a compound, from the initial candidate selection to preparation of the compound library and posterior evaluation and final selection of the most promising candidates in terms of selectivity, serum stability, and BBB-transport.
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