Antibody Screening Using a Human iPSC-based Blood-Brain Barrier Model Identifies Antibodies that Accumulate in the CNS

Drug delivery across the blood-brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood-borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor-mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off-target effects, driving the search for new and potentially improved brain targeting reagent-RMT pairs. To this end, we deployed human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells as a model BBB substrate on which to mine for new RMT-targeting antibody pairs. A nonimmune, human single-chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC-derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1-scFv, exhibiting a 26-fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1-scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor-mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain

A novel approach for the fabrication of polymer bonded magnetic materials - permanent magnets, with the use of microwave radiation

85 σ.Οι μαγνήτες σπανίων γαιών Νεοδυμίου – Σιδήρου - Βορίου (Nd2Fe14B ), αποτελούν τους ισχυρότερους μόνιμους μαγνήτες που έχουν παρασκευασθεί και συνεπώς χρησιμοποιούνται σε πληθώρα εφαρμογών. Οι μαγνήτες αυτοί χρησιμοποιούνται ευρέως σε μορφή όπου η μαγνητική σκόνη περιβάλλεται από μία πολυμερική μήτρα η οποία λειτουργεί σαν το συνδετικό μέσο το οποίο προσφέρει τόσο συνεκτικότητα και μηχανικές αντοχές στο τελικό υλικό, όσο και προστασία από οξείδωση στην μαγνητική σκόνη. Μέχρι στιγμής, οι τεχνικές οι οποίες χρησιμοποιούνται για την μορφοποίηση του τελικού προϊόντος είναι συμβατικές τεχνικές μορφοποίησης πολυμερών και εμφανίζουν ορισμένα μειονεκτήματα όπως σχετικά μεγάλους χρόνους επεξεργασίας και αδυναμία παρασκευής πολύπλοκων αντικειμένων μεγάλου όγκου. Στην εργασία αυτή, πραγματοποιήθηκε σύνθεση μαγνητών δέσμιων με πολυμερές υλικό με μια νέα τεχνική. Η επεξεργασία των δειγμάτων έγινε με μικροκύματα, μέσω της οποίας αναμένεται να ξεπεραστούν τα προβλήματα των συμβατικών μεθόδων. Σαν συνδετικό μέσο επιλέχτηκε εποξειδική ρητίνη (DGEBA), ένα ευρέως χρησιμοποιούμενο συνδετικό μέσο. Στην εργασία έγιναν δοκιμές για την παρασκευή μαγνητικών υλικών σε συμβατικούς φούρνους μικροκυμάτων για διαφορετικές περιεκτικότητες του τελικού προϊόντος σε μαγνητική σκόνη. Με την τεχνική με χρήση μικροκυμάτων που ακολουθήσαμε, επιτύχαμε παρασκευή μαγνητικών υλικών με συνδετικό υλικό πολυμερές με ιδιότητες, ανάλογες με αυτές που παρουσιάζονται στη βιβλιογραφία.Neodymium – Boron – Iron rare earth permanent magnets are the strongest type of manufactured permanent magnets. These magnets are widely used in many modern applications. Moreover, these magnets are used in a polymer bonded form, where the polymer matrix provides both mechanical strength and corrosion protection to the final product. Conventional casting methods are currently applied in order to shape the final product. These methods exhibit drawbacks such as a lengthy processing time, as well as an inability to produce larger objects with a more complex shape. In the current project, polymer bonded magnets were produced utilizing a novel technique. The samples were processed by microwave radiation in order to overcome problems associated with conventional methods. DGEBA epoxy resin, a commonly used binder, was used. The experimental approach consisted of producing permanent magnets with different magnetic content in conventional microwave ovens. The final products exhibit properties in accordance with bibliographic references.Λουκάς Ι. Γούλατη

Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

<div><p>The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.</p></div

Endocytosis of scFvs into SK-BR-3 cells and quantification of intracellular accumulation.

<p>(a) Immunolabeling of surface and internalized scFvs. Soluble scFvs were dimerized via their <i>c-myc</i> epitope tags and pulsed onto SK-BR-3 cells at 37°C for 2 hours to allow for internalization. Fluorophores with different emission spectra were used to immunolabel surface-bound scFv (Alexa647, pseudo-colored pink) and, after permeabilization, intracellular scFv (Alexa488, pseudo-colored green). Nuclei were visualized with DAPI (pseudo-colored blue). Arrowheads indicate the distinct pattern of internalized scFv M16. Scale bar is 5μm. (b) Quantification of scFv association with SK-BR-3 cells. scFv pre-dimerized with 9E10-Alexa488 was dosed onto live SK-BR-3 cells and allowed to traffic at 37°C for 2 hours (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145820#sec002" target="_blank">Materials and Methods</a> for assay details) and total cell-associated scFv assayed by flow cytometry. Internalized scFv was also quantified by flow cytometry after removal of the cell surface bound scFv by trypsinization. Total cell-associated scFv is normalized to H7 scFv and internalized and surface scFv sum to the totals for each clone. (n = 8 for H7 and M16 and n = 12 for N5, ***, p < 0.001). (c) Quantification of scFv internalization after pre-treatment with endosomal acidification inhibitor, BafA1. Internalized scFv was normalized to that for wild-type H7 in the absence of BafA1 treatment (n = 6 for H7, M16 and N5, ***p<0.001).</p

Intracellular co-localization of scFvs with endosomal and lysosomal markers.

<p>(a) SK-BR-3 cells which had been allowed to endocytose scFv dimers (green in merged) were counterstained with an antibody against early endosome antigen type 1 (EEA1, red in merged). (b and c) The same steps were used to counterstain with antibodies against lysosomal-associated membrane proteins 1 and 2 (LAMP1 and LAMP2, red in merged). As indicated by arrowheads, all scFvs co-localized with EEA1, LAMP1 and LAMP2. Scale bar is 5μm. (d) Co-localization with EEA1, LAMP1, and LAMP2 was quantified by Pearson correlation coefficient (**, p <0.01, *, p <0.05).</p

Quantitative analysis of scFvs isolated from the CDRH1his library using yeast surface display.

<p>(a) Fraction TfR bound to M mutants after 10 minute incubation at pH 5.5. (n = 5 for M mutants and n = 13 for H7, and ***, p <0.001) (b) Fraction TfR bound to N and 5P mutants after 10 minute incubation at pH 5.5. (n = 8 for N and 5P mutants and n = 13 for H7 mutant as in panel a) and ***, p <0.001 *, p <0.05). (c) Apparent equilibrium binding affinity of select clones on the surface of yeast at pH 7.4. Mean data from five independent experiments are plotted along with the fitted equilibrium binding isotherms. The legend shows numeric values for the best-fit equilibrium binding affinity (K_d) and associated 95% CI.</p

Creation and screening methodology for a histidine-saturated CDRH1 library based on the wild-type anti-TfR scFv, H7.

<p>(a) The pESO-H7sdm vector contained a mutant scFv H7, that was specially designed to harbor a unique restriction site (<i>SpeI</i>) followed by two stop codons in CDRH1. <i>SpeI</i> digestion of pESO-H7sdm produced a linearized backbone that would be undetectable by flow cytometry if introduced as a reclosed vector into yeast (no c-<i>myc</i> epitope tag expression as a result of the double stop codon). A double-stranded DNA cassette containing the histidine-saturated CDRH1 was built using two cycles of PCR, from primers and a degenerate oligonucleotide (IP90T). Although depicted as a single entity for simplicity, IP90T was in fact a mixture of millions of unique ssDNA oligos representing all possible combinations of histidine in CDRH1. The CDRH1his cassette and linearized pESO-H7sdm were used to create the CDRH1his library by homologous recombination in yeast. (b) Screening and assessment of yeast-displayed scFvs was accomplished by saturating with recombinant human transferrin receptor (rhTfR) followed by a 10 minute incubation in acidic buffer (pH 4.0–6.5) and assay for TfR dissociation. A pH 5.5 buffer was used to simulate endosomal conditions relevant to transferrin-TfR dissociation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145820#pone.0145820.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145820#pone.0145820.ref020" target="_blank">20</a>]. A dissociation control was included by using a pH 7.4 buffer as opposed to acidic buffer at this step. The dissociation process was halted by addition of an excess volume of ice cold pH 7.4 buffer. Surface constructs were subsequently immunolabeled and analyzed by flow cytometry. The histogram depicts the response of yeast-displayed wild-type scFv H7 to 10 minute incubation with buffers having acidic pH.</p

Analysis of soluble M16, N5 and H7 scFvs.

<p>(a) Magnetic bead assay to determine the pH-sensitivity of TfR-binding using soluble protein. Soluble scFvs were captured on the bead surface via their <i>c-myc</i> epitope tags and incubated with rhTfR. After 10 minute incubation in pH 7.4 or pH 5.5 buffer, the fraction of TfR bound at pH 5.5 versus pH 7.4 was assayed by flow cytometry. (n = 10 for M16, n = 14 for H7 and n = 10 for N5, ***, p <0.001, *, p <0.05) (b) Whole-cell immunolabeling using monomeric scFvs and artificial scFv dimers formed via the scFv <i>c-myc</i> epitopes as described in Materials and Methods. Proteins were allowed to traffic for 2 hours in SK-BR-3 cells at 37°C. Meta-z stacks were captured and recombined into a maximum intensity z-projection to better visualize surface versus intracellular protein. Scale bars = 5μm.</p