Naturally occurring antibodies isolated from PD patients inhibit synuclein seeding in vitro and recognize Lewy pathology

Whole blood (100 ml) from 25 clinically diagnosed PD patients (aged 50–65) was purchased from Sanguine Biosciences. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved as previously described [30]. Briefly, the cells were isolated on Ficoll-Paque Plus (GE healthcare) and cryopreserved in 90% FBS and 10% DMSO.

To screen and clone naturally occurring human mAbs to α-synuclein protein, a panel of 7 peptides covering the center region and C-terminus of α-synuclein (amino acids 61–140) were designed and synthesized (Table S1). The peptides included phosphorylation at Ser-129 and Ser-87 and truncation at amino acid positions 110 and 120. The peptides were synthesized by solid-phase chemistry and their purity was confirmed to be higher than 95% by LC–MS (New England Peptide, Inc. and Eton Bioscience, Inc.). Biotinylated peptides were synthesized by connecting biotin moieties via a LC linker to either the N- or C- terminus of the peptides.

Full-length (140 amino acids) human α-synuclein gene SNCA was synthesized at Genewiz Inc and cloned in an pUC57 entry vector. The Avi-α-synuclein sequence was codon optimized for bacterial expression. Xbal and NotI sites were introduced by PCR using Phusion High Fidelity PCR Master Mix (Thermo Fisher); the PCR products were double digested (NEB), gel purified, and ligated into pET28 vector following the manufacturer’s protocol to generate His–Thrombin–Avi-tagged full-length protein. Human SNCA was PCR amplified from a pUC57 vector (Genewiz Inc) with primers (Eton Bioscience) overlapping with SNCA and pcDNA2004 vector and 3′ primers with sequences encoding either -V5 or -HA tag. The fragments were subsequently gel purified and assembled into the vector using a Gibson Assembly Cloning Kit (NEB) following the manufacturer’s protocol. The products were transformed into DH5α competent cells (Thermo Fisher) and the plasmids were confirmed by sequencing. To generate a negative control for the seeding assay, an α-synuclein recombinant version lacking amino acids 60–93 was generated. Primers were designed to amplify two fragments (1–59 and 94–140) of α-synuclein. Q5 high fidelity master mix (NEB) was used for PCR (12 cycles) using pcDNA2004 vector with inserts of SNCA-V5 or SNCA-HA as template. The resulting PCR product was treated with DpnI (NEB) and then transformed into DH5α competent cells (Thermo Fisher), and colonies were sequenced to confirm the correct deletion (amino acid 61–92 deletion). Finally, the expression of the α-synuclein with the different tags was confirmed in HEK293 cells by Western blot of cell lysate samples.

Full-length wild-type α-synuclein, with a C-terminal Avi-tag, thrombin cleavage site and His-tag, was produced in E.coli BL21 (DE3) (Thermo Fisher) cells in a 10-L wave bag. 3 h after induction with IPTG (Sigma-Aldrich), the cells were harvested, and the pellets were stored at − 80 °C. The pellets were re-suspended and thawed in lysis buffer (BugBuster Master mix, Millipore) with protease inhibitor cocktail (cOmplete Ultra EDTA free, Roche). The suspension was centrifuged, and the supernatant was heated for 1 h at 60 °C; then centrifuged at 5250 x g at 4 °C for 30  min. The supernatant was buffer exchanged to 50 mM Bicine pH 8.3. Size-exclusion chromatography with multi-angle static light scattering (SEC–MALS) analysis was used to estimate the total amount of α-synuclein. The required amounts of BirA enzyme, biotin, ATP and magnesium acetate were added for biotinylation overnight per manufacturer instructions (BirA biotin–protein ligase bulk reaction kit, Avidity LLC). Biotinylation was confirmed by SEC–MALS analysis of biotin–synuclein binding to streptavidin-PE. The material was applied to a His-tag resin (Roche Complete His-tag purification Resin), washed three times to remove impurities and the α-synuclein was eluted by thrombin during overnight incubation in 200 mM Tris-HCl, 1.5 M NaCl, 25 mM CaCl2, pH 8.4 and purified on a Superdex 75 SEC column. The protein was quantified by absorption measurements at 280 nm using an extinction coefficient of 0.59 ml mg⁻¹ cm⁻¹. The high purity of the biotinylated α-synuclein was confirmed by SDS-PAGE and analytical SEC (Fig. S1a and b). In addition, purified and biotinylated α-synuclein was mixed with streptavidin-PE and analyzed by SEC–MALS, showing that all α-synuclein was indeed biotinylated (Fig. S1c). The reactivity of biotinylated α-synuclein was assessed by ELISA using a streptavidin-coated plate (described below). The protein is fully reactive to antibodies Syn303 (Biolegend) and C20 (Santa Cruz Biotechnology), which recognize N-terminal (amino acids 1–5) and C-terminal (amino acids 120–140) of synuclein, respectively.

Synuclein peptide and protein baits were prepared by mixing biotinylated peptides or proteins with streptavidin-APC or streptavidin-PE (Thermo Fisher). The majority of peptides and free biotin (a negative control) were prepared at a 1:9 ratio (SA:peptide), incubated for 15 min on ice and passed over a BioSpin 30 column (Biorad) to remove free peptide. The full-length protein and the aggregation prone C-terminally biotinylated peptide 61–95 were prepared at a 1:4 ratio and were used without column clean-up. Each tetramer was used at a final concentration of 36 nM, based on the streptavidin concentration (Table S1). Identification of antigen-specific, memory B cells was performed as previously described [30]. Briefly, PBMCs from 4–6 PD donors were thawed and rested overnight in complete RPMI media (RPMI with 10% FBS and 1% pen/strep) at 37 °C. The B cells were enriched by positive selection with CD22+ magnetic beads (Miltenyi Biotec). Cells were labeled at a final concentration of 20 million per ml in FACS buffer [Tris-buffered saline (TBS) at pH 7.4, with 2 mM EDTA and 0.25% bovine serum albumin (BSA), Fraction V] with the extracellular markers IgG-FITC, CD19-PerCPCy5.5, and CD27-PECy7 (all from BD Biosciences) and the dual-labeled protein/peptide tetramers. To determine nonspecific binding of the tetramers, antibody-labeled cells were incubated with the biotin tetramers, used at the concentration of the pool of peptides (for 8 peptides = 288 nM). The cells and peptides were incubated for 1 h at 4 °C with gentle mixing. After washing, the cells were resuspended at 20 x 10⁶/ml in FACS buffer. The live/dead marker DAPI (Thermo Fisher) was added before the cells were sorted on a Beckman Coulter MoFlo XDP. The gates were set using the negative control and the CD19+, IgG+, CD27^hi, and antigen double-positive live cells were collected by single-cell sorting directly into PCR plates containing cold RT-PCR reaction buffer and RNaseOUT (Thermofisher). Plates were centrifuged briefly and stored at − 80 °C.

Heavy- and light-chain (HC/LC) antibody variable regions were recovered using a two-step PCR as described previously [30]. Briefly, cDNA of variable chain fragments was synthesized using Superscript III First Strand Synthesis Kit (Thermo Fisher), then amplified by nested PCR using pooled forward primers for leader sequence and reverse primers specific to Cγ, Cκ, and Cλ. The step II PCR fragments were subsequently linked via overlap extension PCR followed by digestion of the product with XbaI and XhoI (New England Biolabs). The digested product was subsequently cloned into a dual-CMV-based human IgG1 mammalian expression vector.

Cloned mAbs were transiently transfected into Expi293™ cells (Thermo Fisher) and media were harvested by centrifugation at 72 h post transfection. The IgG was purified from the culture media by Protein A affinity chromatography as previously described [30]. The IgGs were quantified by UV–VIS absorption at 280 nm, and the quality of IgGs was examined by SEC–MALS to assess the monomer content and the amount of aggregates and SDS-PAGE under reducing and non-reducing conditions to assess purity. All IgGs employed in our studies were more than 98% monomeric.

Monomeric, full-length α-synuclein, generated as described above, was aggregated by incubation for 5–6 days at 37 °C in a rotator in the presence of small Teflon beads (1/16”). The samples were centrifuged for 15 min at 20,000 g to separate monomers/oligomers and aggregates. The pellet was stored at − 80 °C to be used as bait and the supernatant was injected on SEC–MALS to quantify the α-synuclein content in the pellets.

Antibody-conjugated beads were prepared following Schrum et al. [33]. Briefly, carboxyl groups on CML Latex Beads (Sigma) were activated with EDAC (50 mg/ml) dissolved in MES coupling buffer (50 mM MES pH 6.0, 1 mM EDTA). Mouse monoclonal anti-V5 antibody (Sigma) in phosphate-buffered saline (PBS) was added to the beads with shaking for 3–4 h, and then washed for later use. HEK293 cells (ATCC, less than 30 passages) were plated (20,000 per well) in a 96-well plate (Costar) in DMEM high-glucose media (Cellgro) supplemented with 10% FBS (Gibco), 1% penicillin, 1% streptomycin and 1% l-glutamine (Hyclone) and left overnight at 37 °C in 8% CO₂. The cells were then transfected using FuGENE HD (Promega). Briefly, 50 ng of Syn-HA and 50 ng Syn-V5 plasmids (or Syn-HA with negative control plasmid pcDNA-SNCA-Del61-92-V5) and 0.3 μl of FuGENE were mixed, incubated for 10 min, and then 10 μl of complexes were incubated with cells at 37 °C for 24 h.

Synuclein aggregates were thawed at room temperature for 15 min, followed by vortexing and dilution to 1 mg/ml. 4 μg of aggregates and 200 μg of anti-synuclein antibodies were mixed to a final volume of 50 μl in PBS (Gibco), incubated for 2 h at 37 °C with shaking and diluted with 350 μl of medium. Finally, 100 μl of the mixture was added to each of four wells and incubated for 72 h. Control antibodies used included mouse Syn211 which prevented synuclein PFF uptake and cell-to-cell transmission of pathology [39] (positive control, ThermoFisher), mouse anti-FLAG M2 (isotype control, Sigma) and human anti-RSV antibody (human isotype control). Cells were detached by adding 50 μl of 0.25% Trypsin-EDTA PBS (Gibco). Then, 150 μl of media was added, and the cells were collected by centrifugation. The cells were lysed in 100 μl of ice-cold Lysis Buffer [1% Triton-X (Sigma) in TBS (Quality Biological)] supplemented with protease inhibitors (Roche) on ice. The lysates were centrifuged to remove cell debris (3000 x g, 5 min, at 4 °C) and 80 μl of supernatant was transferred into a cold 96-well round bottom plate. 150,000 beads in 10 μl (TBS + 1% Triton X-100 and 1X protease inhibitors) were added to each well and incubated overnight at 4 °C with shaking at 750 rpm on a Microplate Genie (USA Scientific). The next day, the beads were centrifuged and washed twice in 200 μl ice-cold Post-IP Buffer (0.2% TritonX-100 in Lysis Buffer).

The capture beads were washed two times in ice-cold FCM staining buffer (BD) by centrifugation. Mouse anti-HA-SureLight APC antibody (Columbia Biosciences) was added to each sample and incubated 40–60 min at 4 °C. The samples were washed three times in FCM Staining Buffer, resuspended in 200 μl FCM staining buffer, and flow cytometry analysis was performed using the MACSQuant (Miltenyi Biotec). The percent positive signal was calculated as the number of APC-positive particles divided by the total number of particles and normalized against that of samples incubated with seeds only (no antibody).

Pierce streptavidin-coated 96-well plates or Costar high-binding plates were coated with the individual biotinylated synuclein peptides (400 nM final) or control (bovine actin, 1 μg/ml) diluted in TBS overnight at 4 °C, respectively. The IgG concentration of the antibodies was determined by Octet with Protein A biosensors using a Protein A calibrator set (ForteBio). The anti-synuclein IgGs, diluted to 10 µg/ml in TBS-T (TBS containing 0.05% Tween 20 and 0.25% BSA), were added to the blocked wells in duplicate and incubated at room temperature for 2 h. After washing, goat-anti human IgG Fab-HRP (1:2000) or goat anti-mouse-HRP (1:4000, Jackson Labs) was added and incubated for 1 h. Plates were washed five times with TBS-T and developed with 100 μl SureBlue Reserve TMB Microwell Peroxidase Substrate (KPL). The reaction was stopped by the addition of 100 μl of TMB stop solution (KPL), and the absorbance at 450 nm was measured using a Tecan M1000 plate reader. Antigen-specific binding was defined as an OD450 greater than 0.5 and at least threefold above the secondary antibody alone. To confirm these results, the antibodies that met the criteria for antigen specificity were serially diluted fivefold in TBST from a starting concentration of 10 μg/ml and retested against the antigen for which they demonstrated reactivity.

The relative binding of the antibodies to full-length synuclein was assessed by biolayer interferometry measurements (Octet Red 384, ForteBio) [8]. Biotinylated synuclein protein was immobilized on Streptavidin (SA) Dip and Read biosensors for kinetics (ForteBio). Real-time binding curves were measured by applying the sensor in a solution containing 100-nM antibody. To induce dissociation, the biosensor containing the antibody–synuclein complex was immersed in assay buffer without antibody. The immobilization of synuclein to sensors, the association and the dissociation steps, were followed in different ionic strength buffers containing 10% FortéBio kinetics buffer as assay buffer. The relative association and dissociation kinetic curves were compared to qualitatively assess the efficiency of antibody binding to peptides encompassing different synuclein epitopes.

The affinities of antibodies for synuclein peptides were determined in solution on a MicroCal Auto-iTC200 system (Malvern). Synuclein peptides at 40 μM were titrated in 20 steps of 2 μl per step, in identical buffers containing 200 μM aSyn-323.1, aSyn-336.1 and aSyn-338.1, respectively. The thermodynamic parameters and the equilibrium dissociation constants, Kd, were determined upon fitting the ITC data to a model assuming a single set of binding sites corresponding to an antibody:synuclein = 1:2 binding model.

Post-mortem human brain tissue was obtained from the VU University Medical Center. Sections (5-µm thick) from formalin-fixed paraffin-embedded PD brain tissue (mesencephalon containing the substantia nigra pars compacta) were mounted on coated glass slides (Menzel gläser superfrost plus, VWR international) and dried overnight at 37 °C. Slides were deparaffinized in xylene and rehydrated through descending alcohol concentrations. Endogenous peroxidase activity was blocked by incubating the slides for 30 min in PBS (pH 7.4) containing 0.3% H₂O₂. Before immunodetection, sections were untreated, treated with formic acid for 30 min at RT or autoclaved for 20 min in 10 mM citric acid pH6.0. Between incubation steps, sections were rinsed in PBS. All antibodies were diluted in antibody diluent (Immunologic) and incubated overnight at RT. Human anti-α-synuclein antibodies aSyn-323.1, aSyn-336.1 and aSyn-338.1 were used at a concentration of 0.5 μg/ml. Mouse anti-alpha-synuclein antibody LB509 (Thermofisher) was used at a concentration of 1.25 μg/ml. Primary antibodies were detected with goat-anti-human-HRP (dilution 1:250, 60 min at RT, Santa Cruz) or goat-anti-mouse/rabbit-HRP (ready-to-use, 30 min at RT, EnVision Dako). To visualize the staining 3,3′-diaminobenzidine (DAB; Dako) was used. Slides were counterstained with haematoxylin, dehydrated and mounted with Quick-D mounting medium (Klinipath). Different antibodies were assessed in serial tissue sections.