Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

  1. Iga Kucharska
  2. Elaine Thai
  3. Ananya Srivastava
  4. John L Rubinstein
  5. Régis Pomès
  6. Jean-Philippe Julien  Is a corresponding author
  1. Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Canada
  2. Department of Biochemistry, University of Toronto, Canada
  3. Department of Medical Biophysics, University of Toronto, Canada
  4. Department of Immunology, University of Toronto, Canada
5 figures, 3 tables and 4 additional files

Figures

Figure 1 with 2 supplements
Comparison of PfCSP and PbCSP repeat sequences and structures.

(A) Schematic representations of PfCSP strain NF54 and PbCSP strain ANKA, each comprising an N-terminal domain, central repeat region, and C-terminal domain. The junctional region (J) immediately following the N-terminal domain of PfCSP is indicated. Colored bars represent each repeat motif. The sequences of each CSP central repeat region and corresponding peptides used in the study are shown below their respective schematics. (B-G) Conformational ensembles of CSP peptides in solution from molecular dynamics simulations. (B) Superposition of the conformations of the four PfCSP-derived peptides at each nanosecond. The peptides are aligned to the conformational median structure and only the backbone is shown for clarity. (C) Ensemble-averaged backbone-backbone hydrogen-bonding maps for each PfCSP peptide sequence. The propensity for hydrogen bonds between the NH groups (y-axis) and CO groups (x-axis) is indicated by the color scale on the right. (D) Sample molecular dynamics snapshots of the highest-propensity turn for each PfCSP peptide are shown as sticks with hydrogen bonds shown as gray lines. The highest-propensity turn for each peptide is indicated by the arrowhead on the corresponding hydrogen-bonding map. (E) Superposition of the conformations of the four PbCSP-derived peptides at each nanosecond. The peptides are aligned to the conformational median structure and only the backbone is shown for clarity. (F) Ensemble-averaged backbone-backbone hydrogen-bonding maps for each PbCSP peptide sequence. The propensity for hydrogen bonds between the NH groups (y-axis) and CO groups (x-axis) is indicated by the color scale on the right. (G) Sample molecular dynamics snapshots of the highest-propensity turn for each PbCSP peptide are shown as sticks with hydrogen bonds shown as gray lines. The highest-propensity turn for each peptide is indicated by the arrowhead on the corresponding hydrogen-bonding map.

Figure 1—figure supplement 1
Ensemble-averaged hydrogen-bonding propensities for PfCSP- and PbCSP-derived peptides.

(A) The propensity for hydrogen bonds between the NH groups of the backbone (y-axis) and CO groups of the side chains (x-axis) is indicated by the color scale on the right. (B) The propensity for hydrogen bonds between the NH groups of the side chains (y-axis) and CO groups of the backbone (x-axis) is indicated by the color scale on the right. (C) The propensity for hydrogen bonds between the NH groups of the side chains (y-axis) and CO groups of the side chains (x-axis) is indicated by the color scale on the right.

Figure 1—figure supplement 2
Experimental details of MD simulations.

(A) Time evolution of the radius of gyration of the different peptides from MD simulations. Shading represents the standard error of the mean computed from the different simulation repeats (see Materials and methods). The simulations are statistically converged after 100 ns. The average radius of gyration for each peptide is reported on the right. The individual peptides have been shifted on the y-axis to allow for ease of visualization. (B) The simulated propensity of having a specific hydrogen bond is shown on the diagonal, with the propensity of two specific hydrogen bonds (P(A∩B)) shown above the diagonal. Each individual NPNA motif is labelled from I-V. The difference of the simulated values from the calculated values (P(A)·P(B)) is shown below the diagonal. These differences are all well below the average standard error of mean of 0.04, confirming that the individual motifs are uncorrelated. (C) A mathematical example showing that the β-turn and pseudo β-turn propensities are independent and uncorrelated (P(A∩B)=P(A)·P(B)). This holds true for all the simulated peptides.

Biophysical characterization of 3D11 Fab-PbCSP binding.

(A) Binding kinetics of twofold dilutions of 3D11 Fab to PbCSP. Representative sensorgrams are shown in black and 2:1 model best fits in red. Data are representative of three independent measurements. (B) Isothermal titration calorimetry (ITC) analysis of 3D11 Fab binding to PfCSP at 37°C. Above, raw data of PbCSP (0.005 mM) in the sample cell titrated with 3D11 Fab (0.4 mM). Below, plot and trendline of heat of injectant corresponding to the raw data. KD and N values resulting from three independent experiments are indicated. Standard error values are reported as standard error of the mean (SEM). (C) Results from size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) for the 3D11 Fab-PbCSP complex. A representative measurement of the molar mass of the 3D11 Fab-PbCSP complex is shown as the red line. Mean molar mass and standard deviation are as indicated. (D) SDS-PAGE analysis of resulting Peaks 1 and 2 from SEC-MALS. Each peak was sampled in reducing and non-reducing conditions as indicated by + and -, respectively.

Figure 3 with 2 supplements
3D11 Fab binding to PbCSP repeat peptides.

(A) Affinities of 3D11 Fab for PAPP, NAND, NPND, and Mixed peptides as measured by ITC. Symbols represent independent measurements. Mean KD values are shown above the corresponding bar. Error bars represent SEM. Peptide sequences are as indicated to the right of the plot, with variable residues underlined and shaded residues indicating those resolved in the corresponding X-ray crystal structures. (B) The 3D11 Fab binds the PAPP (pink), NAND (purple), NPND (blue) and Mixed (red) peptides in nearly identical conformations. mAb 3D11 CDRs are indicated. (C) Overview and side view of the NAND peptide (purple) in the binding groove of the 3D11 Fab shown as surface representation (H-chain shown in black and K-chain shown in gray). (D) Van der Waals interactions formed by side chain atoms of both Ala and Pro residues are indicated by orange dashed lines, and those unique to Pro6 and Pro10 are indicated by green dashed lines.

Figure 3—figure supplement 1
Experimental details of mAb 3D11 binding.

(A) 3D11 Fab binding to peptides representative of the PbCSP αTSR domain (residue 263–318; red) and the full C-terminal domain (residues 202–318; PbC-CSP; blue). PbCSP (residue 24–318) was used as a positive control (black). (B) Composite omit map electron density contoured at 1.0 sigma (blue mesh) around PbCSP peptides PAPP, NAND, NPND and Mixed in complex with the 3D11 Fab. (C) Slight differences in H-bonding at the N- and C-terminal ends of the PbCSP repeat peptides when bound to the 3D11 Fab in the crystal structures. PbCSP peptides are colored as in Figure 3. Antibody residues partaking in H-bonds are colored orange. mAb 3D11 HCDR2 is colored in black and KCDR2 in white.

Figure 3—figure supplement 2
Interactions between mAb 3D11 aromatic side chains and PbCSP peptides.

Interactions formed between PbCSP peptide residues and aromatic side chains of the 3D11 Fab HCDR (black) and KCDR (white). PbCSP peptides are colored as in Figure 3.

Figure 4 with 4 supplements
Spiral organization of the PbCSP repeat upon 3D11 Fab binding.

(A) The cryoEM map of the 3D11 Fab-PbCSP complex reveals high-resolution information for seven predominant 3D11 Fabs. Regions corresponding to Fabs are colored from pink to gray. (B) CryoEM map of the 3D11 Fab-PbCSP complex is shown as a transparent light gray surface with the PbCSP region highlighted in black. (C) The PbCSP model built into the cryoEM map is shown in dark gray as sticks and aligned to the schematic representation of the PbCSP protein sequence.

Figure 4—figure supplement 1
CryoEM data processing workflow in cryoSPARC v2.
Figure 4—figure supplement 2
CryoEM analysis of the 3D11 Fab-PbCSP complex.

(A) Left panel – a representative cryoEM micrograph used to calculate the 3D11 Fab-PbCSP cryoEM map. Right panel – a cryoEM micrograph of the 3D11 Fab-PbCSP complex from a 200 kV screening microscope with individual particles highlighted with white circles. Scale bars: 50 nm. (B) Selected 2D class averages of the 3D11 Fab-PbCSP complex. (C) Particle orientation distribution plot. (D) Fourier shell correlation curve from the final 3D non-uniform refinement of the 3D11 Fab-PbCSP complex in cryoSPARC v2. (E) Local resolution (Å) plotted on the surface of the cryoEM map. (F) Low-pass filtered (20 Å) cryoEM map of the 3D11 Fab-PbCSP complex with observable 3D11 Fabs numbered. (G) CryoEM map of PbCSP (gray mesh) with the model shown as sticks (black carbons).

Figure 4—figure supplement 3
Comparison between the 3D11 Fab-PbCSP cryoEM structure and 3D11 Fab-NPND peptide crystal structure.

(A) Color representation of the backbone RMSD of the 3D11 Fab variable region and PbCSP core epitope between the 3D11 Fab-PbCSP cryoEM structure and the 3D11 Fab-NPND peptide crystal structure. (B) Color representation of the all-atom RMSD of the 3D11 Fab residues engaging in Fab-Fab contacts between the 3D11 Fab-PbCSP cryoEM structure and the 3D11 Fab-NPND peptide crystal structure. RMSD values were calculated using PyMOL (Schrodinger LLC, 2015) and plotted by color on the secondary structure of the 3D11 Fab-NPND peptide crystal structure.

Figure 4—video 1
3D Variability Analysis on 165,747 particle images of the 3D11 Fab-PbCSP complex.
Figure 5 with 3 supplements
Homotypic interactions between 3D11 Fabs stabilize the 3D11 Fab-PbCSP complex.

(A and B) Close-up views of adjacent 3D11 Fabs from the cryoEM structure in complex with PbCSP (black). 3D11 Fabs bound to PbCSP form homotypic contacts with each adjacent Fab through two interfaces; one consisting of CDRs from the heavy and light chains of Fabs A and B (interface 1, A), and the second mediated by residues in FR3 of Fab A HC and FR3 of Fab C LC (interface 2, B). Variable domains of Fabs are shown in white. HCDR1, −2,–3, and KCDR1, −2 and −3 are colored yellow, orange, red, green, blue and purple, respectively. Residues forming Fab-Fab contacts are labeled with the position of the Fab in the cryoEM model (A, B or C) indicated in subscript. mAb 3D11 affinity-matured residues that engage in Fab-Fab contacts, but do not directly interact with PbCSP are highlighted in yellow with red font. Black dashed lines denote H-bonds. (C) Sequence alignment of mAb 3D11 with its inferred germline precursor. INT1 and INT2 refer to the two interfaces shown in (A) and (B). Green highlight: germline-encoded residues involved in homotypic interactions; Red: affinity-matured residues involved in homotypic interactions; Yellow highlight: affinity-matured residues involved in homotypic interactions that do not directly interact with PbCSP. (D) Binding affinity of WT 3D11 and H-58/73 germline-reverted mutant (Mut) Fabs to NPNDx1 (gray bars) and NPNDx2 (white bars) peptides as measured by ITC. Symbols represent independent measurements. Mean KD values resulting from at least two independent experiments are shown. Error bars represent standard error of the mean. An unpaired one-tailed t-test was performed using GraphPad Prism 8 to evaluate statistical significance: *p<0.05.

Figure 5—figure supplement 1
Homotypic contacts between 3D11 Fabs in the 3D11 Fab-PbCSP cryoEM structure.

(A) Main interaction interfaces between adjacent 3D11 Fabs in the 3D11 Fab-PbCSP cryoEM structure. (B) Table of contacts between 3D11 Fabs. HB: hydrogen bond (3.8 Å cut-off).

Figure 5—figure supplement 2
Negative-stain EM analysis of 3D11 IgG-PbCSP complexes.

(A) SEC chromatogram of 3D11 IgG-PbCSP complexes. (B) Representative negative-stain (NS) micrographs of particles present in selected SEC fractions: 1 – soluble aggregates, 2 - 3D11 IgG-PbCSP complexes, 3 – unbound 3D11 IgGs. Scale bars: 100 nm. (C) Comparison of NS 2D class averages of the 3D11 IgG-PbCSP complex (upper panels) to 2D class averages of the 3D11 Fab-PbCSP complex (lower panels).

Figure 5—figure supplement 3
Comparison between cryoEM structures of 3D11 Fab-PbCSP and 311 Fab-PfCSP (PDB ID: 6MB3) (Oyen et al., 2018).

CryoEM maps of the 3D11 Fab-PbCSP (A and E) and 311 Fab-PfCSP (C and G) complexes are shown as a transparent light gray surface with the CSP density highlighted in black for PbCSP and in blue for PfCSP. The CSP models built into the cryoEM maps are shown as gray for PbCSP; (B and F) or blue for PfCSP; (D) and H) sticks and aligned to the schematic representations of their respective protein sequences.

Tables

Table 1
X-ray crystallography data collection and refinement statistics.

Despite binding in nearly identical conformations, differences exist in the molecular details of 3D11 Fab binding to each peptide that provide key insights into mAb 3D11 recognition of PbCSP. Our crystal structures revealed that more van der Waals contacts were formed by a Pro residue in the PPPP and NPND motifs compared to an Ala at the same position in the PAPP and NAND motifs (Figure 3D). Consequently, the epitopes of the NAND, NPND and Mixed peptides had a slightly greater buried surface area (BSA; 753, 762, and 765 Å2, respectively) than the PAPP peptide (743 Å2), which only consists of Ala-containing motifs (Supplementary file 2). In particular, Pro10 of the PPPP motif found in the NAND and NPND peptides forms more van der Waals interactions with antibody residues H.Asn33 and H.Tyr52 compared to Ala10 of the PAPP motif present in PAPP and Mixed peptides. Similarly, Pro6 of the NPND motif in the NPND and Mixed peptides makes additional interactions with antibody residue K.Leu50 that are not present for Ala6 of the NAND motif within the PAPP and NAND peptides (Supplementary file 2). These differences in interactions observed at the atomic level directly relate to the binding affinities measured by ITC, where the PbCSP peptides that bury more surface area in the 3D11 paratope have the highest binding affinities (Figure 3A).

3D11-PAPP3D11-NAND3D11-NPND3D11-Mixed
BeamlineAPS-23-ID-DAPS-23-ID-DNSLS-II-17-ID-1APS-23-ID-B
Wavelength (Å)1.0331701.0332000.9793291.033167
Space groupP3221P3221P3221P3221
Cell dimensions
a,b,c (Å)59.3, 59.3, 233.559.7, 59.7, 234.959.9, 59.9, 235.060.3, 60.3, 233.7
α, β, γ (°)90, 90, 12090, 90, 12090, 90, 12090, 90, 120
Resolution (Å)*40.0–1.60 (1.70–1.60)40.0–1.55 (1.65–1.55)40.0–2.27 (2.37–2.27)40.0–1.55 (1.65–1.55)
No. molecules in ASU1111
No. observations1,210,903 (196,555)684,564 (117,091)450,057 (47,142)1,423,235 (247,601)
No. unique observations64,371 (10,497)70,664 (11,753)23,398 (2,556)72,981 (12,222)
Multiplicity18.8 (18.7)9.5 (9.7)19.1 (17.4)19.5 (20.3)
Rmerge (%)10.3 (84.7)8.4 (80.1)13.8 (57.1)8.3 (78.0)
Rpim (%)2.4 (20.1)2.9 (26.5)3.2 (13.5)1.9 (17.6)
<I/σ I>16.3 (1.5)13.8 (1.5)19.0 (4.1)19.6 (1.7)
CC½99.9 (68.0)99.9 (56.7)99.9 (93.5)99.9 (84.3)
Completeness (%)99.9 (100.0)98.3 (97.2)99.3 (94.4)100.0 (100.0)
Refinement Statistics
Reflections used in refinement64,27570,66023,32772,843
Reflections used for R-free1999198611732000
Non-hydrogen atoms3823391536653858
Macromolecule3411342333823439
Water384380259359
Heteroatom281122460
Rwork§/Rfree15.9/18.816.4/18.416.6/22.216.6/18.1
Rms deviations from ideality
Bond lengths (Å)0.0160.0100.0060.011
Bond angle (°)1.431.150.871.22
Ramachandran plot
Favored regions (%)98.998.097.798.2
Allowed regions (%)1.12.02.31.8
B-factors (Å2)
Wilson B-value27.124.032.026.3
Average B-factors35.031.435.231.2
Average macromolecule33.629.434.829.7
Average heteroatom54.454.854.457.6
Average water molecule46.341.938.341.2
  1. * Values in parentheses refer to the highest resolution bin.

    † Rmerge = Σhkl Σi | Ihkl, i - < Ihkl > | / Σhkl < Ihkl > .

  2. ‡ Rpim = Σhkl [1/(N – 1)]1/2 Σi | Ihkl, i - < Ihkl > | / Σhkl < Ihkl > .

    § Rwork = (Σ | |Fo | − |Fc | |) / (Σ | |Fo |) - for all data except as indicated in footnote ¶.

  3. ¶ 5% of data were used for the Rfree calculation.

Table 2
CryoEM data collection and refinement statistics.
Data collection
Electron microscopeTitan Krios G3
CameraFalcon 3EC
Voltage (kV)300
Nominal magnification75,000
Calibrated physical pixel size (Å)1.06
Total exposure (e- /Å2)42.7
Number of frames30
Image processing
Motion correction softwarecryoSPARCv2
CTF estimation softwarecryoSPARCv2
Particle selection softwarecryoSPARCv2
3D map classification and refinement softwarecryoSPARCv2
Micrographs used2080
Particles selected669,223
Global resolution (Å)3.2
Particles contributing to final map165,747
Model building
Modeling softwareCoot, phenix.real_space_refine
Number of residues built3085
RMS (bonds)0.002
RMS (angles)0.56
Ramachandran favored (%)95.8
Rotamer outliers (%)0.5
Clashscore6.27
MolProbity score1.63
EMRinger score2.54
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
 Recombinant DNA reagentpcDNA3.4-3D11 Fab HC (plasmid)This paperN/A3D11 Fab heavy chain gene in pcDNA3.4 TOPO vector
 Recombinant DNA reagentpcDNA3.4-3D11 Fab 58/73 HC (plasmid)This paperN/A3D11 Fab germline-reverted mutant heavy chain gene in pcDNA3.4 TOPO vector
 Recombinant DNA reagentpcDNA3.4-3D11 Fab KC (plasmid)This paperN/A3D11 Fab light chain gene in pcDNA3.4 TOPO vector
 Recombinant DNA reagentpcDNA3.4-PbCSP-6xHis (plasmid)This paperN/APbCSP gene with His tag in pcDNA3.4 TOPO vector
 Recombinant DNA reagentpcDNA3.4-PbC-CSP-6xHis (plasmid)This paperN/APbC-CSP gene with His tag in pcDNA3.4 TOPO vector
 Recombinant DNA reagentpcDNA3.4-PbCSP-αTSR-6xHis (plasmid)This paperN/APbCSP αTSR gene with His tag in pcDNA3.4 TOPO vector
 Cell line (Homo sapiens)FreeStyle 293 F cellsThermo Fisher ScientificCat# R79007
 Cell line (Mus musculus)3D11 hybridoma cell lineYoshida et al., 1980BEI Resources #MRA-100; RRID:AB_2650479
 Chemical compoundGIBCO FreeStyle 293 Expression MediumThermo Fisher ScientificCat# 12338026
 Chemical compoundGIBCO Hybridoma-SFMThermo Fisher ScientificCat# 12045076
 Chemical compoundFectoPRO DNA Transfection ReagentVWRCat# 10118–444
 Chemical compoundFetal bovine serumThermo Fisher ScientificCat# 12483–020
 Antibody3D11 IgG (mouse monoclonal)Yoshida et al., 1980N/APurified from 3D11 hybridoma cell line; See Materials and methods
 Recombinant protein3D11 FabThis paperN/ASee Materials and methods for concentrations and masses used, and buffer conditions
 Recombinant protein3D11 Fab H-58/73This paperN/ASee Materials and methods for concentrations and masses used, and buffer conditions
 Recombinant proteinPbCSPThis paperN/ASee Materials and methods for concentrations and masses used, and buffer conditions
 PeptidePAPP (PAPPNANDPAPPNAND)This paperN/ADerived from PbCSP repeat region
 PeptideNAND (PPPPNANDPPPPNAND)This paperN/ADerived from PbCSP repeat region
 PeptideNPND (PPPPNPNDPPPPNPND)This paperN/ADerived from PbCSP repeat region
 PeptideMixed (PPPPNPNDPAPPNAND)This paperN/ADerived from PbCSP repeat region
 PeptideNPNDx1 (PPPPNPNDPPPP)This paperN/ADerived from PbCSP repeat region
 PeptideNPNDx2 (PPPPNPNDPPPPNPNDPPPPNPND)This paperN/ADerived from PbCSP repeat region
 Software, algorithmGROMACS 5.1.4Abraham et al., 2015; Berendsen et al., 1995http://manual.gromacs.org/documentation/5.1.4/; RRID:SCR_014565
 Software, algorithmLINCSHess et al., 1997; Hess, 2008N/A
 Software, algorithmParticle-Mesh Ewald algorithmDarden et al., 1993; Essmann et al., 1995N/A
 Software, algorithmNosé-Hoover thermostatNosé, 1984; Hoover, 1985N/A
 Software, algorithmParrinello-Rahman algorithmParrinello and Rahman, 1981N/A
 Software, algorithmVMDHumphrey et al., 1996https://www.ks.uiuc.edu/Research/vmd/; RRID:SCR_001820
 Software, algorithmMatplotlibHunter, 2007https://matplotlib.org/; RRID:SCR_008624
 Software, algorithmOctet Data Analysis Software 9.0.0.6ForteBiohttps://www.fortebio.com/products/octet-systems-software
 Software, algorithmMicroCal ITC Origin 7.0 Analysis SoftwareMalvernhttps://www.malvernpanalytical.com/
 Software, algorithmASTRAWyatthttps://www.wyatt.com/products/software/astra.html; RRID:SCR_016255
 Software, algorithmGraphPad Prism 8GraphPad Softwarehttps://www.graphpad.com/; RRID:SCR_002798
 Software, algorithmEPUThermoFisher Scientifichttps://www.fei.com/software/
 Software, algorithmSBGridSBGrid Consortiumhttps://sbgrid.org/; RRID:SCR_003511
 Software, algorithmcryoSPARC v2Punjani et al., 2017https://cryosparc.com/
 Software, algorithmPhenix (phenix.refine; phenix.real_space_refine)Adams et al., 2010https://www.phenix-online.org/; RRID:SCR_014224
 Software, algorithmUCSF ChimeraPettersen et al., 2004https://www.cgl.ucsf.edu/chimera/; RRID:SCR_004097
 Software, algorithmUCSF ChimeraXGoddard et al., 2018https://www.cgl.ucsf.edu/chimerax/; RRID:SCR_015872
 Software, algorithmCootEmsley et al., 2010https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/; RRID:SCR_014222
 Software, algorithmPyMOLThe PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.https://pymol.org/2/#products; RRID:SCR_000305
 Software, algorithmPDBePISAKrissinel and Henrick, 2007https://www.ebi.ac.uk/pdbe/pisa/; RRID:SCR_015749
 OtherHomemade holey gold gridsMarr et al., 2014N/A
 OtherHomemade carbon gridsBooth et al., 2011N/A

Additional files

Supplementary file 1

Hydrogen-bonding propensities from simulations of peptides in solution.

(A) Hydrogen-bonding propensity for each simulated motif and lifetime of each β-turn for the four PfCSP-derived peptides. (B) Hydrogen-bonding propensity for each simulated motif and lifetime of each β-turn for the four PbCSP-derived peptides.

https://cdn.elifesciences.org/articles/59018/elife-59018-supp1-v1.docx
Supplementary file 2

Table of contacts between 3D11 Fab and PbCSP peptides.

Rows are shaded according to the number of times interactions are observed between all four crystal structures, summed in the final column.

https://cdn.elifesciences.org/articles/59018/elife-59018-supp2-v1.docx
Supplementary file 3

Table of contacts between one of the 3D11 Fabs and PbCSP in the cryoEM.

https://cdn.elifesciences.org/articles/59018/elife-59018-supp3-v1.docx
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  1. Iga Kucharska
  2. Elaine Thai
  3. Ananya Srivastava
  4. John L Rubinstein
  5. Régis Pomès
  6. Jean-Philippe Julien
(2020)
Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11
eLife 9:e59018.
https://doi.org/10.7554/eLife.59018