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.

incubated for 3 h in the cocktail cleavage.Then, the solvent was evaporated applying an argon current.Diethyl ether was added at 0C to precipitate the peptide and eliminate non-peptidic impurities, and the mixture was centrifuged (4 o C, 5000g, 5 min).The supernatant liquid was decanted and the process was repeated three times.After that, the cleaved peptides were dissolved in H2O/MeCN (1:1, 0.1% TFA) and filtered off the resin.Finally, peptides were lyophilised.

Conjugation of peptides to sulfo-cyanine5 N-hydroxysuccinimide (sCy5-NHS) ester
After peptide cleavage and cyclization, the conjugation to sCy5-NHS fluorophore was performed in anhydrous medium to avoid the hydrolysis the NHS moiety. 1 eq. of the crude peptide was treated with 1.5 eq. of fluorophore and 4 eq. of DIPEA in anhydrous DMSO.The mixture was allowed to react for 30 min and the completion of the reaction was assessed by HPLC and MALDI-TOF.Ethanolamine was used to quench the unreacted esters.

Purification of peptides
Peptides were dissolved in H2O/MeCN (the percentage depending on the peptide) and filtered through 0.45 μm filters.Then, they were purified on an Agilent 1260 Infinity II system with ChromScope software, a 1260 VWD, a 1260 Preparative Binary Pump, a 1260 Column Organizer and a 1290 Preparative Fraction Collector.An Aeris Peptide XB-C18 100 LC Column (250 x 10 mm, 5 m, Phenomenex) was used, with MeCN (0.1% TFA) and H2O (0.1% TFA) as solvents and a flow rate of 12 mL/min.The fractions containing the peptide were analysed by MALDI-TOF, pooled together and lyophilised.

Peptide stability in human serum
Peptides were dissolved in human serum at a final concentration of 100 µM and then incubated at 37 o C. Aliquots of 50 µL were taken at different times (0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 24 h and 48 h) and flash frozen with acetone/solid CO2.Samples were treated with 200 µL of MeOH to precipitate serum proteins.Samples for UHPLC analysis were centrifuged and filtered through a 0.22 µm filter before injection.Aliquots were analysed with UHPLC-UV (flow = 0.61 mL/min; gradient 5-95% MeCN (0.036% TFA) in water (0.045% TFA) in 2 minutes).The amount of peptide remaining was calculated by comparing the area under the curve of the several samples.

Protein production and conjugation
Expression of the Cys-GFP E. coli BL21 cells were transfected by heat shock with pET29 plasmid carrying the gene for GFP expressing an extra cysteine at the N-terminus.One single colony was used to inoculate 500 mL of LB media, containing kanamycin (50 µg/mL) and the culture was grown overnight at 150 rpm at 37 o C. Cells were harvested by centrifugation at 6000 g for 15 min at 4 °C.Pellets were resuspended in lysis buffer (50 mM TRIS-HCl pH=8.0, 150 mM NaCl, 1% Triton X-100 and 1 mM PMSF) and intracellular content was released by sonication at 40% amplitude, with 25 s ON and 30 s OFF pulses, for a total of 10 min on ice.Lysis suspension was clarified centrifuging at 25000 g for 25 min at 4 °C.The protein was purified by Immobilised Metal Chelate Affinity Chromatography (1 mL IMAC HisTrap™ HP column from Cytiva) on a fast protein liquid chromatography system (FPLC, BIO-RAD NGC).Fractions containing the protein were pooled.

Expression of the anti-CD133 scFv antibody
The production of the anti-CD133 scFv antibody was performed following an adapted version of the protocol described in Swaminathan et al. 2013. 2 The plasmid encoding the scFv, supplied by Prof. Jayanth Panyam (University of Minesotta), was modified to incorporate a Sortase A recognition sequence at the C-terminal and a His12 tag.

Sortase A ligation of the BCN linker to the scFv
Once the antibody was produced, the reaction with Sortase A (SrtA, BPS Bioscience Catalog #71046, or in-house produced from Addgene Catalog #75144) to remove the His-tag and incorporate the BCN linker (Gly-PEG3-endo-BCN, BroadPharm, Catalog #BP-24232) was performed.5 eq. of SrtA and 10 eq. of BCN linker were added to 1 eq. of scFv (35 µM) in 50 mM Tris-HCl, 150 mM NaCl and 0.5 mM CaCl2.The reaction was allowed to proceed for 2 h at 22C.Then, SrtA and the unreacted antibody were purified from the conjugate in an IMAC nickel-loaded column, where the BCN-modified antibody eluted in the flow-through.The purity of the product was assessed by SDS-PAGE and LC-QTOF.

Conjugation of the peptides to Cys-GFP
The conjugation of GFP incorporating an extra cysteine with the maleimido-labelled peptide was accomplished by mixing the two components in a 1:25 ratio in PBS buffer.They were left to react for 2h, and the progress of the reaction was assessed by LC-MS and SDS-PAGE.

Conjugation of the peptides to the anti-CD133 scFv antibody
The conjugation of the anti-CD133 scFv antibody incorporating the BCN moiety (previously labelled with the fluorophore) with the azide-labelled peptide was accomplished by mixing the two components in a 1:25 ratio in PBS buffer.They were left to react for 1h, and the progress of the reaction was assessed by SDS-PAGE.

Labelling of anti-CD133 scFv antibody with sulfo-Cyanine5 NHS ester
One eq. of anti-CD133 antibody (50 µM stock concentration) in PBS was treated with 10 eq. of sCy5 NHS ester (10 mM stock concentration in DMSO).The mixture was allowed to react for 60 min and the excess fluorophore was removed by dialysis (Slide-A-Lyzer™ MINI Dialysis Devices, 2K MWCO, ThermoFisher) against PBS following the manufacturer's protocol (dialysing for 15 min and changing the buffer four times).The correct conjugation was assessed by SDS-PAGE and LC-QTOF.

SDS-PAGE
SDS-PAGE electrophoresis was carried out using BioRad system (Miniprotean cell) in a 15% Tris gel using the following running buffer: 1.92 M Gly, 0.25 M Tris, 1% SDS, pH 8.3.The gel run at 180 V until the marker reached the bottom of the gel.Protein molecular weights were approximated by comparison to a protein marker (Precision Plus Protein Dual Color Standards from BioRad).Proteins were visualized by Coomassie staining (staining solution: 10% AcOH, 0.25 g brilliant blue; unstaining solution: 40% MeOH, 10% AcOH in water).

LC-QTOF
Samples were analysed using the Q-TOF X500B connected to an Exion LCAD chromatograph.The peptide analysis was performed using a column Acquity BEH C18 (50 x 2 mm x 1.7 μm).applying a 5-95% gradient MeCN (0.1% FA) in water (0.1% FA) in 5 min.Q-TOF MS was operated in the positive ion mode for the analysis.The mass spectra were recorded across the range of 100-3000 Da with a fixed collision energy of 10 V. Data acquisition and evaluation was performed using SCIOX OS software.

Cell culture
HeLa cells were cultured in DMEM media (1000 mg/L glucose) supplemented with 10% fetal bovine serum (FBS), 1% of 2 mM L-glutamine and 1% antibiotics (100 units/mL of penicillin and 100 µg/mL of streptomycin).Cells were expanded at 80% confluence and grown at 37C with 5% CO2.All work performed with human cells follow the ethical principles and EU Directive 2004/23/EC.

Cell transfection of HeLa cells to express higher levels of TfR1
In a 96-well plate, 10,000 HeLa cells per well were seeded and left to grow for 24 h.A mixture of Lipofectamine 3000 (0.1 µL/well, Lipofectamine™ 3000 Transfection Reagent, ThermoFisher Scientific), P3000 Reagent (0.2 µL/well) and the DNA pTfR-mNeonGreen (Plasmid #129608, Addgene) (100 ng/well) in Opti-MEM medium was prepared.The mixture was incubated for 15 min, added to the cells, and incubated for 48 h.

Cell binding assays
48-hour post-transfection, 50 μl of the sulfo-Cy5-labelled peptides were added to each well at the appropriate concentration (from 5 to 600 nM) and incubated at 4C for 1 hour.Cells were washed three times with 100 μL of PBS, trypsinized with 25 μl of trypsin for 5 min at 37C and, then 75 μL of supplemented medium with formalin (33% v/v) was added.Cells were detached by repeated pipetting and analysed using an Agilent NovoCyteflow cytometer (Agilent).sCy5 was analysed at 640 nm while mNeon-Green at 488 nm.Data was analysed by plotting the geometric mean vs the concentration of the peptide.

Permeability assays in the in vitro BBB cellular model
These experiments were performed using the model developed by Cecchelli and collaborators. 3Briefly, endothelial cells derived from pluripotent stem cells (brain like endothelial cells) and bovine pericytes were defrosted in gelatin-coated Petri dishes (Corning).Pericytes were cultured in DMEM pH 6.8 while endothelial cells were cultured in supplemented endothelial cell growth medium (sECM) (Sciencells).After 48 h, endothelial cells were seeded in 0.4 µm polycarbonate 12-well Transwell inserts (8,000 cell/well) and pericytes were plated in 12-well plates (50,000 cells/well) previously coated with Matrigel and gelatin, respectively.The medium was changed every 2-3 days and the assays were performed 7-8 days after seeding by placing the transwells into new wells without pericytes.100 nM of protein or antibody conjugates was used in each well.Lucifer Yellow (50 µM) was added as a control of barrier integrity (Papp < 17•10 -6 cm/s).To perform the assay, 500 L of the compound in Ringer HEPES was added to the donor compartment and 1500 L of Ringer HEPES was introduced into the acceptor compartment.The plates were incubated for 2 h at 37 o C, and the solutions from both compartments were recovered and analysed.The samples were evaluated in triplicates.The amount of protein conjugates was quantified by fluorescence with Fluorescence Spectrophotometer F2500 HITACHI.The selected slit for excitation and emission was 5 nm and 10 nm, respectively.The voltage for the detector was 700 V. Apparent permeability was calculated with the following formula: where Papp is obtained in cm/s, QA(t) is the amount of compound at the time t in the acceptor well, VD is the volume in the donor well, t is time in seconds, A is the area of the membrane in cm and QD(t0) is the amount of compound in the donor compartment at the beginning of the experiment.
Experiments were designed and executed from the Octet BLI Discovery 13.0 software.The biotinylated human Transferrin Receptor (TFR-H82E5, Acro Biosystems) was immobilized onto the biosensor by dipping the sensors into a solution of 1 µg/mL hTfR for 500 s, followed by dipping the sensor into a fresh solution of binding buffer to stablish a baseline for 200 s.
Titrations were executed at 25ºC while rotating at 1000 rpm.In the association phase, the sensors were dipped into a solution containing the peptides at several concentrations in binding buffer for 150 s.After reaching equilibrium, the biosensors were dipped into fresh binding solution to monitor the dissociation kinetics for 200 s.Kinetic data were collected and processed using a 1:1 binding model to obtain the affinity constants using the Octet Analysis Studio 13.0.

Statistical analysis
Unpaired two-tailed student t tests were applied to evaluate the significant difference or p values between data sets using Prism 6.0c software.

Figure S1 Continued Figure S1
Figure S1 Continued

Figure S2
Figure S2Circular dichroism spectra of peptides.

Figure S3 CFigure S4 Figure S5
Figure S3Mass spectra of GY1 peptide incubated in human serum at different time points.

Figure S6
Figure S6 SDS-PAGE characterization of antibody conjugates with the peptide shuttle nonmodified (A) and modified with sulfo-cyanine-5 (B).(C) SDS-PAGE characterization of GFP protein conjugation with the peptide shuttle.

Figure S7
Figure S7 Characterization of GFP conjugate mass.Raw data (left) and deconvoluted mass spectra (right).

Table S1
Sequence, molecular formula, molecular weight (MW), UPLC characterisation (tR, retention time) and purity after synthesis and purification of peptides

Table S2
BBB-model permeabilities.All values are Mean SD