Dendritic Cell‐Mediated Cross‐Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS‐CoV‐2

Abstract Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS‐CoV‐2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross‐priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT, are first generated by grafting an anti‐RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo‐EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high‐avidity neutralizing interaction. Then, by C‐terminal fusion of an anti‐DNGR‐1 scFv to TNT, the bispecific trimerbody TNTDNGR‐1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross‐priming. Therapeutic administration of TNTDNGR‐1, but not TNT, protects K18‐hACE2 mice from a lethal SARS‐CoV‐2 infection, boosting virus‐specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus‐neutralizing antibody activity and demonstrate the therapeutic potential of the Fc‐free strategy that can be used advantageously to provide both immediate and long‐term protection against SARS‐CoV‐2 and other viral infections.


Figure S1 .
Figure S1.Purified TN tandem and TN T trimerbody recognize efficiently and similarly SARS-CoV-2 S protein RBD from different viral strains.A) SDS-PAGE analysis in reducing conditions of purified TN and TN T .Both antibodies were purified from HEK-293 conditioned medium.B) SEC-MALS data proving monomeric (TN, top) and trimeric (TN T , bottom) states of each antibody.The black lines correspond to the differential refractive index (left axis) and the red line to the measured molar mass (right axis).Asterisk (*) in TN T chromatogram indicates an experimental artifact spike cause by a chromatography pause.C) TN (top) and TN T (bottom) far-UV CD spectra.D) TN (top) and TN T (bottom) thermal denaturation profiles.E) TN (50, 10, 1 nM) and TN T (10, 1, 0.1 nM) binding activity against immobilized B.1, beta, gamma, kappa and delta RBD proteins.ACE2 (0.1 μg/mL) interaction with different RBD strains was included as an inner control (left panel).All assays were performed by triplicate.Independent and mean values ± SEM are plotted.The binding kinetics of the TN and TN T to B.1 (F) and B.1.351(beta)(G) RBD were measured, using the antibodies at 50 and 10 nM, with 0.5

Figure S2 .
Figure S2.Microfluidic diffusional sizing (MDS) analysis and cryo-EM data processing and reconstruction of HexaPro S protein/TN T complex.MDS was used to measure the change in hydrodynamic radius (Rh) of TN T trimerbody and its subsequent complex with SARS-CoV-2 S protein.A) Plot of the Rh versus the number of residues in the polypeptide chain.The values of the hydrodynamic radii displayed in this graph correspond to the Rh values reported in the literature for a range of native folded proteins (red) and highly denatured polypeptide chains (blue) obtained using dynamic light scattering and pulsed field gradient nuclear magnetic resonance.TN T and S protein/TN T complex Rhs (green) correlate with the expected for folded proteins with their nominal number of amino acids.B) Charts indicating the number of residues and Rh of folded (red) and unfolded (blue) polypeptides and proteins used in this correlation according to literature.Number of residues of TN T , 1077, and S protein/TN T complex, 4941, trimeric conformations in solution (green).C-H, cryo-EM data processing and reconstruction of HexaPro S protein/TN T complex.C) Regarding Cryo-EM analyses top panel shows an example of motion corrected micrograph.D) Subset of 7 2D classes obtained from the 2D classification job run after particle picking.E) Electron density maps representative of final 3D complex reconstruction at 3.8Å and local map resolution.Three volume subclasses were identified as the majoritarian ones.F) Viewing Direction Distribution map illustrating how many images have a viewing direction at each (elevation, azimuth) bin.G) Validation and resolution estimation results.Fourier Shell correlation (FSC) curves are measured between half-sets of particles that are used to compute two half-map reconstructions.FSC curves using a mask (green, red, purple) improved the resolution estimate of 3.8Å versus 4.5Å for conventional refinement.Notably, a local focused refinement using the same mask is unable to improve resolution beyond the conventional refinement result due to the flexibility of the region.H) Cryo-EM data collecting and analysis table.

Figure
Figure S3.S protein/TN T complex model shows TN T hexavalent binding to S protein causing the 3-up RBD conformation with one-RBD position distortion.Side (A) and top views (B) of the HexaPro S protein/TN T complex model showing TN T embracing the S protein in the 3-up RBD prefusion conformation.As shown, TN T binds simultaneously and in a biparatopic manner, both E VHH and V VHH, to the three S protein RBDs.S protein subunits are colored in yellow, steel blue and olive green, while E VHH and V VHH are in purple and magenta, respectively.C) Top view of the alignment of the HexaPro S protein/TN T model (S protein in pale yellow, E VHH in dark blue and V VHH in magenta) and 7B18 PDB model (S protein in aquamarine, E VHH in steel blue and V VHH light pink)showing how the binding of the VHHs to their respective RBDs occurs in both structures.While in two of them the positions are conserved, TN T presence causes a shift in the third RBD location respect to the 7B18 model, even though the manner that both VHHs recognize this RBD domain is preserved.For easy viewing just the three-RBDs from the S trimer are showed.

Figure S4 .
Figure S4.Purified TN T DNGR-1 performs as globular bispecific trimerbody in solution targeting viral antigens to DNGR-1-expressing DCs in vitro and boosting systemic S-specific responses in vivo.A) Coomassie blue staining after SDS-PAGE analysis in reducing conditions comparing purified samples of both TN T and TN T DNR-

Figure S5 .
Figure S5.Absorbance measurements for serum antibodies binding to SARS-CoV-2 antigens.Data showing S-specific IgM (A), and S-(B), RBD-(C) and N-specific (D) total IgG quantification on day 5 p.c. Measurements for S-(E), RBD-(F) and N-specific (G) total IgG quantification on day 20 p.c.All graphs represent mean ± SEM (n=4

Figure S6 .
Figure S6.Efficacy experiment mice serum analysis for SARS-CoV-2 neutralization.Neutralization experiments for live SARS-CoV-2 (B.1 strain) virus using pooled mice sera from each group (n=4 mice/group) and obtained on 5 (A) and 20 (B) days p.c. NT50 is represented as a dotted line.C) Calculation of administrated antibodies (sotrovimab, TN T and TN T DNGR-1) concentration present in serum at 5-and 20-days p.c.All graphs represent mean ± SEM for a single experiment.All measurements were done by duplicate.

Figure S7 .
Figure S7.Flow cytometry analysis showing TN T DNGR-1 treatment of SARS-CoV-2 infection induced S-specific CD8 + T cell responses in the lungs.A) Representative gating for S-Tet staining of CD8 + T cells obtained from processed K18-hACE2 mice lungs at day 20 p.c. Single and live CD8 + T cells were gated from lymphocyte morphology cloud as CD45 + , CD11c-, CD8 + , CD4 -.B) CD44 + S-Tet + dot plot representation from processed lung CD8 + T cells at day 20 p.c. showing exemplary mice from the different treatment groups (PBS, sotrovimab and TN T DNGR-1).For ICS data analysis CD4 + (C) and CD8 + (D) T cells were gated from single and live lymphocyte morphology cloud as CD3 + and CD4 + or CD8 + .Herein an exemplary ICS gating for CD107a, IFN-γ, TNF-α and IL-2 is represented as paired dot plot.E) ICS for CD107a, IFN-γ, TNF-α or IL-2, represented as frequency of CD4 + (left panel) and CD8 + (right panel) responding cells in the lungs at 20 days p.c. for each individual marker.All graphs represent individual dots and mean ± SEM (n=4 mice/group) for a single experiment.