Protein expression and purification

Expression and purification of LCB1-Bn and LCB1-ABD-Bn were performed as previously described⁴¹ with slight modifications. E. coli BL21(DE3), transformed with the corresponding plasmid containing the Barstar gene under its natural promoter, was grown in the ZYM-5052 autoinduction medium supplemented with kanamycin (50 mg/L) at 25 °C on a shaker in 2.5 L flasks containing 300 ml medium. The biomass was collected and resuspended in a buffer containing 100 mM Tris-HCl, 250 mM sucrose, 0.5 mM EDTA, pH 8.0, and 0.5 mM PMSF and lysozyme was added to a final concentration of 30 µg/ml. After 30 min, the cell suspension was sonicated. The resulting cell lysate was centrifuged and the supernatant containing the soluble fraction was collected and filtered through a 0.22 μm membrane. The buffer was applied to a 5 ml HisTrap HP column (Cytiva) equilibrated with 10 CV of buffer A (20 mM NaPi, pH 7.5, 500 mM NaCl) with 30 mM imidazole at 5 ml/min. The column was washed with 10 CV of buffer A containing 30 mM imidazole at a rate of 5 ml/min. To wash away Barstar from the Bn: Barstar complex immobilized on the column, the column was washed with 25 CV of buffer A with 6 M guanidine hydrochloride at a 1 mL/min rate. Following this, target proteins free from the Barstar inhibitor were renatured directly on the column by a linear gradient (60 CV) of guanidine hydrochloride 6 M–0 M at a 1 mL/min rate. After the renaturation step, the column was washed with 10 volumes of solution B (20 mM NaPi, pH 7.5, and 100 mM NaCl), and the target protein was eluted from the column with buffer B containing 250 mM imidazole. The eluate obtained after affinity chromatography was diluted with buffer C (20 mM NaPi and pH 7.5) by a factor of 5 and applied on a 5 ml HiTrap Q HP column (Cytiva), equilibrated with 10 volumes buffer C at 5 ml/min. The column was washed with 10 volumes of buffer C, and the target protein was eluted at a 5 mL/min rate with a linear gradient of NaCl (0–500 mM) in buffer C. The volume of the gradient was 20 CV. The fractions containing the target proteins were pooled and sterilized by filtration through a 0.22 μm membrane. Protein concentration was measured by A₂₈₀ with its corresponding E_0.1% (Figure S1, Supporting Information), and endotoxins were assayed using the LAL gel-clot test Pyrotel (G5003, Associates of Cape Cod, Inc.) with levels below 20 EU/mg. Barnase activity was assessed as previously described⁴³.

Trimeric S protein and human ACE2 extracellular domains were expressed in suspension HEK293 using PEI-mediated transient transfection and purified from culture medium via IMAC followed by gel filtration on a Superdex S200 column (Cytiva). Both constructs featured the C-terminal 6xHis tag. The S protein sequence included HexaPro stabilizing mutations as previously described⁴⁴.

SPR

Assay SPR analyses were performed using Biacore X100 and Biacore 8 K optical biosensors (GE Heathcare, USA) with standard CM5 sensor chips (Cytiva, USA) at 25 °C. PBS buffer (Cytiva, USA) was used as the running buffer.

Albumins (HSA, BSA, and MSA) and S protein trimer were immobilized on the CM5 chip surface using a carbodiimide reaction at pH 4.5 (albumins) or 5.0 (Spike). The protein concentration in the immobilization buffer was 50 µg/ml with a contact time of 7 min at a flow rate of 5 µl/min. The average final immobilization levels were 6000 ± 2000 RU and 17,000 ± 3000 RU for albumins and S protein, respectively.

Analyte solutions in PBS were injected at a flow rate of 5 µl/min for 3 min and dissociation was recorded for 5 to 15 min. The concentrations of analyte solutions were 25–400 nM (with S protein ligand) or 100–16,000 nM (with albumin ligands). When registering the interaction of chimeras with albumins, two injections of glycine buffer (pH 2.5) were performed to regenerate the chip surface (30 s, 30 µl/min).

The resulting sensorgrams were processed in the BIAevaluation 4.1.1 software using the 1:1 interaction model with mass transfer or steady-state affinity model for multi-cycle analyses and kinetic titration model for single-cycle analyses or in the Biacore Insight software v. 3.0.11.15423 using the default 1:1 interaction model with the mass transfer or steady-state affinity model for multi-cycle analyses and kinetic titration model for single-cycle analyses. The descriptions of the 1:1 binding and kinetic titration methods (single cycle model) have been detailed previously⁴⁵.

ACE2 competition assay

ACE2 competition assay was performed following our previous method²⁰. Briefly, 100 µl of recombinant trimeric S protein solution at a concentration of 1 µg/ml in PBS was added to the wells of a 96-well MaxiSorp plate (Nunc, Denmark) and adsorbed at 2–8 °C overnight. After blocking at RT for 1 h, samples were prepared in blocking buffer at three dilutions (1:10, 1:50, and 1:250) in triplicate in a separate 96-well plate. Following this, 100 µl of each sample was transferred to Spike-containing wells and incubated at 700 rpm and 37 °C in a thermostated shaker for 30 min. Following incubation, the plate was washed five times. Next, 100 µl of recombinant hACE2-3xFLAG solution (0.2 µg/ml) in blocking buffer was dispensed into each well. After a 30-min incubation, 100 µl of anti-FLAG antibodies conjugated with horseradish peroxidase (Sigma Aldrich, USA, Cat. # A8592), diluted 1:10,000 in blocking buffer, was added to the wells, and the plate was incubated for another 30 min as previously described. After the final wash, 100 µl of TMB substrate solution was added to each well and incubated in the dark for 15 min. The enzymatic reaction was stopped by adding 10% phosphoric acid solution, and the OD₄₅₀ values were measured using a plate spectrophotometer. Curves illustrating the relationship between OD₄₅₀ values and peptide concentration were generated using GraphPad Prism 8 software.

Pseudovirus neutralization assay

Pseudovirus neutralization assays were conducted using recombinant lentiviruses carrying the SARS-CoV-2 S protein and encoding the firefly luciferase (Luc) as previously described with slight modifications⁴⁶. To generate pseudovirus particles, HEK293T cells (ATCC CRL-3216) were seeded in T75 flasks, grown to 50–70% confluence, and transfected with a mixture of plasmids (15 µg pLuc, 15 µg pGAG, 5 µg pRev, and 2 µg SARS-CoV-2 S per flask) using PEI (75 µg per flask) as the transfection reagent. The cells were then incubated at 37 °C under 5% CO₂ for 72 h in DMEM medium with 10% FBS. After 72 h, the culture supernatant was centrifuged first at 150 g and then at 4000 g. The resulting supernatant aliquots were stored at − 80 °C. HEK293T cells carrying surface hACE2 and hTMPRSS2 (in-lab modified ATCC CRL-3216 via lentiviral transduction and FACS) were seeded in white 96-well plates at a density of 2 × 10⁴ cells/well and incubated overnight. Serial dilutions of samples were prepared in DMEM with 10% FBS. Further, diluted samples (5 µl) were mixed with the medium containing pseudoviruses (50 µl) in 96-well plates and incubated at 37 °C and 5% CO₂ for 1 h. Following this, 50 µl of medium was removed from the wells of the plates with HEK293T-ACE2-TMPRSS2 cells, and the cells were infected with virus-sample mixtures (50 µl/well). The inoculated HEK293T-ACE2-TMPRSS2 cells were then incubated at 37 °C and 5% CO₂ for 48 h. All tests were performed in triplicate. After 48 h of incubation, the medium was collected from the wells with cells, 100 µl of lysis buffer (25 mM Tris-phosphate, pH 7.8, 1% Triton X-100, 10% glycerol, 2 mM DTT) was added to the wells, and the plate was incubated at room temperature for 5 min. Following this, 20 µl of Bright-Glo™ Luciferase Assay Substrate (Promega, USA) was added and the luminescence intensities were measured.

Animal studies

All the animal studies were performed after approval from the Institutional Animal Care and Use Committee (IACUC) at the Biological Testing Laboratory of IBCh RAS (protocol #373/202, 30.03.2023). All methods performed in this study were in accordance with the IACUC guidelines and regulation. These guidelines are equivalent to the ARRIVE guidelines and therefore all methods were performed in accordance with the ARRIVE guidelines.

For the virus challenge experiments, C57BL/6J-TgTn(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)Smoc mice (further referred to as CAG-hACE2), (NM-TG-200002, Shanghai Model Organisms Center, PRC) were obtained from the Animal Breeding Facility of IBCh RAS (Pushchino branch). For MTD evaluation, specific pathogen-free outbred ICR male mice aged six weeks were also sourced from this facility. Similarly, specific pathogen-free BALB/c female mice, aged eight weeks, were used for pharmacokinetic evaluation. Mice were obtained from the Bioresource Collection supported by the Ministry of Science and Higher Education of the Russian Federation (Contract # 075-15-2021-1067).

The animals had been acclimated for two weeks prior to experimental procedures and the ones that showed no abnormalities were randomly separated into experimental groups. Mice were kept in two-corridor barrier rooms under a controlled environment, temperature 20–24 °C, relative humidity 30–60%, 12-h light cycle. Animals were group-housed in standard polycarbonate cages type 3 (820 sq. cm, Techniplast s.p.a) on bedding (LIGNOCEL BK 8/15, JRS, Germany) with ad libitum access to feed (Velaz FORTI 1324 Maintenance Diet, Altromin Spezialfutter GmbH & Co, Germany) and filtered tap water. Cages were also supplied with Mouse House™ (Techniplast, Italy) for environmental enrichment.

For live virus protection experiments, mice were infected with 20 µL of PBS containing 10³ virus plaque-forming units of SARS-CoV-2 (strain hCoV-19/Australia/VIC01/2020 (GISAID: EPI_ISL_406844), Pango lineage B, clade O) via intranasal inoculation. The following day, the mice were treated with 20 µL chimeras intranasally or 100 µL intraperitoneally.

For pharmacokinetic studies, mice were intraperitoneally injected with 100 µL of chimera solution in PBS. Subsequently, 20 µL of blood was collected from the retroorbital sinus using syringes pre-filled with 100 µL of PBS + 6 mM EDTA solution at the specified time points. The samples were then gently centrifuged to remove blood cells, and the supernatant was deep-frozen at − 86 °C for further analysis.

For pharmacokinetics assessment, ICR mice (n = 10 or 6 per group) were administered LCB1-ABD-Bn or vehicle (PBS) intraperitoneally on days 0 and 14 at a dose of 2.7 mg/kg. On day 28, animals were humanely euthanized using carbon dioxide (CO₂) in accordance with institutional animal care guidelines. Serum was separated by centrifugation and stored at − 80 °C until analysis.

Viral load quantification

Total RNA was extracted from homogenized lung tissues using the RIBO-prep nucleic acid extraction kit (AmpliSens, Russia) in accordance with the manufacturer’s protocol. Quantification of SARS-CoV-2 RNA was performed using the AmpliSens^® COVID-19-FL diagnostic kit (AmpliSens, Russia), which is based on reverse transcription and real-time PCR (RT-qPCR) with hybridization-fluorescence detection. The assay targets a conserved region of the RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2 and includes both an internal control for RNA extraction efficiency and an amplification control. Quantification was based on the certified synthetic RNA standard provided with the kit.

Cryo-EM sample preparation and data collection

The water-soluble S protein in PBS at a concentration of 2 mg/ml (approximately 14.4 µM) was mixed with LCB1-Bn from the stock (3 mg/ml, 25 mM Tris, 250 mM NaCl) to molar ratio 1:1.5. The same S protein stock was mixed with LCB1-ABD-Bn (4 mg/ml, 25 mM Tris, 250 mM NaCl) to molar ratio 1:1.5 to the final S protein concentration 1.4 mg/ml (approximately 10.1 µM).

The mixtures were incubated at room temperature for 30–40 min and 3 µl of each mixture was applied to glow-discharged Quantifoil R1.2/1.3 200-mesh grids (Quantifoil Micro Tools GmbH, Germany). The samples were blotted using blot force 0 at 100% humidity and 4 °C for 6 s and then plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).

Data were collected using a 300 kV Thermo Fisher Titan Krios electron microscope equipped with Gatan K3 Summit and 20 eV energy filter (CUHK, China). The total amount of movies in both datasets were recorded at a magnification of x105000 with pixel size 0.83 Å/pixel and defocus range from − 0.8 to − 2.5 μm. Total dose of 51.38 electrons/Å2 were distributed over 50 frames in the movie.

Cryo-EM data processing

All movies were obtained and imported into Relion-3.1.3⁴⁷, WARP 1.0.9⁴⁸, and CryoSPARC v.4.2.0³⁴. Motion correction and CTF estimation of raw movies were performed in all these programs (implemented in Relion-3.1.3, MotionCor2⁴⁹, and CTFFIND 4.1⁵⁰). Further, the default neural network in WARP 1.0.9 was used for particle picking. This particle stack was imported in CryoSPARC v.4.2.0, and after several rounds of 2D classification, particles were selected to make templates for template picking in CryoSPARC v.4.2.0³⁴.Template picking allowed us to collect significantly more particles. Approximately 200k particles were chosen in each dataset after several rounds of 2D classification, Ab initio reconstruction with five classes, and heterogeneous refinement with five classes. The particle stack was then exported to Relion 3.1.3⁴⁷ and extracted with a pixel size of 1.58 Å. The selected 3D classification particles were refined with C1 symmetry in Relion-3.1.3⁴⁷ and CryoSPARC v.4.2.0³⁴. To characterize flexibility of the complexes, 3D variability analysis with three models and a low pass of 8 Å was performed. We also performed Hetero Refinement after 3DVA in CryoSPARC v.4.2.0³⁴ with two extreme positions in motion as the initial volumes to avoid invisibility of RBD/LCB1-ABD-Bn and RDB/LCB1-Bn fragments due to tilt fluctuations. Cryo-EM data processing workflows of SARS-CoV-2 S protein complexes with LCB1-Bn and LCB1-ABD-Bn are demonstrated in Figures S8 and S9, Supporting Information.

Models of LCB1-Bn/Spike and LCB1-ABD-Bn/S protein complexes were prepared using the initial model of binding of the S protein of SARS-CoV-2 to the LCB1 peptide (PDB 7JZL)¹³. We tilted RBD/LCB1-Bn and RBD/LCB1-ABD-Bn fragments of the original model to match with the obtained EM densities. All models adjustments were conducted with Chimera v1.15⁵¹ and Coot 0.9.8.93⁵². Visualization and analysis of the structures were performed using Chimera v1.15⁵¹. The statistics for Cryo-EM data collection and structure refinement were performed using Phenix v1.20⁵³ and summarized in Table S2, Supporting Information.

Label-free quantitative chromatography-mass spectrometry-based pharmacokinetics

Sample protein concentration was estimated by measuring the absorbance at 280 nm (assuming E_0.1%=1) and using a BCA assay (Pierce, USA). The volume containing 10 µg of total protein was dried in a SpeedVac system, dissolved in 10 µL of 1% sodium deoxycholate,100 mM Tris, 10 mM TCEP, 20 mM CAA, pH 7.5 buffer. Proteins were denatured, and disulfide bonds were reduced by heating the solution at 85 °C for 10 min. Trypsin (Molecta, Russia) was added at a 1/50 w/w ratio for overnight digestion at 37 °C. Peptides were cleaned up using SDB-RPS StageTips and dried.

LC-MS analysis was performed on an Ultimate 3000 RSLCnano LC System (Thermo Fisher Scientific) connected to an Orbitrap Tribrid Lumos or Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) via a nano-electrospray source. Cleaned up peptide samples were dissolved in 0.1% formic acid (FA) and 2% acetonitrile (ACN) and loaded onto a pre-column (50 × 0.1 mm) custom-packed with Prontosil C18 5 μm sorbent in a loading buffer containing 2% ACN, 98% H₂O, and 0.1% TFA at a flow rate of 5 µL/min for 4 min and separated at room temperature on a fused silica capillary column (300 × 0.1 mm) custom-packed into a pulled emitter capillary with Reprosil PUR C18 AQ 1.9 μm (Dr. Maisch)^54,55. Peptides were separated with a linear gradient of 0.1% FA, 80% ACN (buffer B) in 0.1% FA in water (buffer A) as follows: 3–35% B over 105 min, 35–50% B in 18 min, 50% B for 1 min, 50–99% B in 0.1 min, 99% B for 3 min, and back to 3% B in 0.1 min, at a flow rate of 500 nL/min.

For protein identification and MS1-based quantitation, MS analysis was performed in DDA mode. The following settings were used for Q Exactive Plus mass spectrometer: MS1 scan at 70 K resolution, scan range 350–1600 m/z, 3e6 AGC target with 35 ms max IT. Ten dependent MS2 scans were collected at 17.5 K resolution, 1.4 m/z isolation window with 0.2 isolation offset, HCD fragmentation with 30 nce, and AGC target 1e5 with max IT 100 ms. The following settings were used for Orbitrap Fusion Lumos mass spectrometer: MS1 scan at 120 K resolution, scan range 350–1600 m/z, standard AGC target and auto maxIT for MS1 and MS2 spectra, total cycle time 2 s, MS2 spectra collected at 15 K resolution, 1.2 m/z isolation window, and HCD fragmentation with 30 nce.

Proteins were identified in PEAKS Studio software version 10.0 (Bioinformatics Solutions Inc.)⁵⁶ with the following parameters: Mus musculus Uniprot protein sequence database (dated 28.06.2022), appended with the target protein sequences LCB1-Bn and LCB1-ABD-Bn, fixed modification of Cys by carbamidomethylation, variable modifications including deamidation of Asn/Gln and oxidation of Met, 1% FDR at the PSM level, and trypsin protease specificity with up to two missed cleavage sites. Standard mass tolerance for Orbi-Orbi was used.

DDA-based peptide and protein identification data were used for MS1-based quantitative analysis on the same data with the PEAKS Studio software label-free-quantitation mode. Relative target protein quantitation between samples was estimated as the sum of all its peptide intensities normalized to the total ion current (TIC). Absolute quantitation was calculated from the total protein abundance and TIC and target protein summed intensity was calculated as follows:

where protein intensity was calculated as the sum of the intensities of all assigned peptide IDs, TIC intensity was integrated throughout the entire LC-MS analysis, and 35 mg/mL was taken as the average mouse blood protein concentration.

The presence of common high-abundance blood plasma proteins greatly affects the stability of the signal for low-abundance target peptides in non-targeted survey MS1 DDA spectra, especially at the beginning and end of the pharmacodynamics curve, where the target protein concentration is low. For precise quantitation, DDA analysis results were used to generate a scheduled PRM method for the top 15 PAB-specific peptides. Relative MS2-based quantitation (top three MS2 peaks were used for each peptide) was analyzed in Skyline software⁵⁷. Target peptide MS2 intensities were normalized to several high abundance albumin peptides. The ratio between the LC peak areas for MS1 and MS2 intensities for each peptide was calculated from samples containing the maximum concentrations of target peptides with good MS1 signal stability followed by MS1 area recalculation for each sample from MS2 intensities and absolute protein abundance estimation using the formula above.

Pharmacokinetic parameters were calculated using the R software environment⁵⁸ with the specialized pkr package. Non-compartmental analysis was performed and AUC was calculated using linear interpolation. Pharmacokinetic curves were generated using the ggplot2⁵⁹ and xgxr packages for R.

Immunogenicity assessment

Anti-drug antibody (ADA) and anti-domain titers were assessed by direct ELISA. Briefly, 96-well plates MaxiSorp plate (Nunc, Denmark) were coated with 100 ul of respective protein solution (1 µg/mL in sodium bicarbonate buffer (pH 9.6)) overnight at 4 °C, and then blocked with PBST containing 0.1% BSA (1 h, RT). Serial dilutions of mouse serum were added and incubated for 1 h at 37 °C. After washing, a HRP-conjugated goat anti–mouse IgG (Fc-specific) antibody (A2554, Sigma-Aldrich) at 1:100,000 dilution was applied for 1 h at 37 °C. After a final wash, TMB substrate was added, and the reaction was stopped by adding 10% phosphoric acid solution, and the OD₄₅₀ values were measured using a plate spectrophotometer. Endpoint titers were determined as the highest serum dilution giving an OD₄₅₀ at least three times higher than the corresponding dilution of control serum.

Neutralizing activity of mouse serum antibodies was evaluated in a SARS-CoV-2 pseudovirus assay as described above, with a minor modification. Briefly, serum samples (diluted 1:50) were pre-incubated with 1 nM LCB1-ABD-Bn and pseudoviruses for 1 h before infecting HEK293T–ACE2–TMPRSS2 cells. Infection levels were quantified by measuring luciferase activity 48 h post-infection, and the resulting decrease in LCB1-ABD-Bn efficacy (compared to control wells lacking serum) was expressed as a percentage.

Statistical analysis

All results are presented as mean ± standard deviation (SD). The sample size (n) for each experiment is provided in the figure legends or text. Statistical analyses were performed using GraphPad Prism 9.3 (GraphPad Software, USA). For pairwise comparisons, the Mann–Whitney test was used. For comparisons of three or more groups, the Kruskal–Wallis test was employed, followed by Dunn’s post-hoc test when applicable. Survival curves were compared using the Log-rank (Mantel–Cox) test. A p-value < 0.05 was considered statistically significant, and significance levels are indicated in the figure legends.