PF-07302048: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR-10741, Ver. 2.0

Title: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein(P2 S) as a Vaccine Antigen

090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)

Study Number: N/A

Parent Compound Number(s): PF-07302048

Alternative Compound Identifiers: N/A

Pfizer Discovery Sciences
Eastern Point Road
Groton, CT

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PF-07302048: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR-10741, Ver. 2.0

Title: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein(P2 S) as a Vaccine Antigen

PRINCIPAL INVESTIGATOR: (b) (6)

CONTRIBUTING SCIENTIST(S):090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)(b) (6)

PREPARED BY:(b) (6)APPROVED BY:(b) (6)PFIZER CONFIDENTIAL
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PF-07302048: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR-10741, Ver. 2.0

Title: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein(P2 S) as a Vaccine Antigen

SYNOPSIS

090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)The binding and structural analysis of SARS-CoV-2 P2 S expressed from DNA that encodes
the same amino acid sequence as BNT162b2 RNA indicate that the encoded P2 S antigen
authentically presents the ACE2 binding site and other epitopes targeted by SARS-CoV-2
neutralizing antibodies.

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Vaccine Antigen
VR-VTR-10741, Ver. 2.0

TABLE OF CONTENTSSYNOPSIS.................................................................................................................................3
LIST OF TABLES.....................................................................................................................4
LIST OF FIGURES ...................................................................................................................4
1. OBJECTIVES ........................................................................................................................5
2. INTRODUCTION .................................................................................................................5
3. MATERIALS AND METHODS...........................................................................................6
3.1. Flow Cytometry Analysis of Binding to Cell Surface-Expressed P2 S ....................6
3.2. P2 S Expression and Purification ..............................................................................6
3.3. Binding Kinetics of P2 S to Immobilized Human ACE2 and a Neutralizing
Monoclonal Antibody by Biolayer Interferometry......................................................6
3.4. Cryo-EM of P2 S.......................................................................................................7
4. RESULTS AND DISCUSSION ............................................................................................9090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)

5. CONCLUSION....................................................................................................................12
6. DEVIATIONS .....................................................................................................................12
7. REFERENCES ....................................................................................................................12
LIST OF TABLES
Table 1.

CryoEM Data Collection, 3D Reconstruction and Refinement
Statistics......................................................................................................8

LIST OF FIGURES
Figure 1.

Binding to Cell Surface-Expressed Recombinant P2 S..............................9

Figure 2.

Biolayer Interferometry Sensorgrams for Binding of P2 S to ACE2PD and B38 mAb......................................................................................10

Figure 3.

CryoEM P2 S Structure at 3.29 Å Resolution ..........................................11

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PF-07302048: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR-10741, Ver. 2.0

Title: Structural and Biophysical Characterization of SARS-CoV-2 Spike Glycoprotein(P2 S) as a Vaccine Antigen
Study Number:

N/A

Functional Area:

Medicinal Sciences

Test Facility: Pfizer Discovery Sciences, Eastern Point Road,
Groton, CT
Study/Testing Initiation Date:

07April2020

Study/Testing Completion Date: 19Aug2020

1. OBJECTIVES

090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)The purpose of this study was to express and characterize the vaccine antigen encoded by
BNT162b2.
2. INTRODUCTION
The coronavirus disease 2019 (COVID-19) vaccine (BioNTech code number BNT162, Pfizer
code number PF-07302048) is an investigational vaccine intended to prevent COVID-19,
which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Coronavirus S is a major target of virus neutralizing antibodies and is a key antigen for
vaccine development. S is a transmembrane glycoprotein responsible for receptor
recognition, attachment to the cell, and viral envelope fusion with a host cell membrane
resulting in genome release. While the membrane-proximal S2 is responsible for membranefusion, the membrane-distal S1, with its receptor-binding domain (RBD), recognizes the host
receptor, angiotensin converting enzyme 2 (ACE2) (Zhou et al, 2020). The RBD forms
membrane distal “heads” on the S trimer that are connected to the body by a hinge. In thenative S, the RBD alternates between an open (up) and closed (down) position. Althoughpotent neutralizing epitopes have been described when the RBD is in the “heads down”
closed conformation, the “heads up” receptor accessible conformation exposes a potentiallygreater breadth of neutralizing antibody targets (Brouwer et al, 2020; Liu et al, 2020;
Robbiani et al, 2020).
The glycoprotein encoded by the vaccine candidate BNT162b2 includes two amino acidsubstitutions to proline (P2 S) locking the transmembrane protein in an antigenically optimal
prefusion conformation (Pallesen et al, 2017; Wrapp et al, 2020). The P2 S antigen was
expressed from DNA and characterized for structure and binding to human ACE2 and
SARS-CoV-2 neutralizing antibodies.

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3. MATERIALS AND METHODS
3.1. Flow Cytometry Analysis of Binding to Cell Surface-Expressed P2 S
A modified pcDNA3.1 Zeo (+) construct encoding the P2 S antigen under control of a CAG
promoter was expressed in Expi293F cells as per the supplied Expifectamine 293
Transfection Kit protocol.

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Cells were collected 48 hr post transfection, washed with TBS buffer, and used at 4-5 x 104
for each condition. Cells were incubated for 1 hr at room temperature (RT) in TBS + 4%
BSA + 0.01 mg/mL 7-AAD to detect non-viable cells and with either (i) 1:100 FITC-labeledanti-6xHis plus 10 nM His-tagged human ACE2 peptidase domain (ACE2-PD); (ii) 100 nM
Alexa-488 labeled anti-Rabbit IgG Fab plus either 33 nM anti-SARS-CoV-2 Spike RBD(αRBD, Sino Biological 40592-T62), anti-SARS-Cov-2 Spike S1 (αS1, Sino Biological
40150-R007), or anti-SARS Spike S2 (αS2, Novus NB100-56578); or (iii) 100 nM
Alexa-488 labeled ant-Human IgG Fab plus either 33 nM CR3022 therapeutic antibody(αCR3022) (Yuan et al, 2020), B38 neutralizing antibody (αB38), or H4 neutralizing
antibody (αH4) (Wu et al, 2020). Cells were washed with TBS and then analyzed in a
V-bottom 96-well plate using a Guava EasyCyte HT flow cytometry system. For eachcondition, three replicates were measured with 3000 events collected per replicate.
3.2. P2 S Expression and Purification
To express SARS-CoV-2 P2 S encoded by BNT162b2 for biophysical characterization, agene encoding the full length SARS-CoV-2 spike (GenBank: MN908947) with two prolines
substituted at residues 986 and 987 (K986P and V987P) followed with a C-terminal HRV3C
protease site and a TwinStrep tag was cloned into a modified pcDNA3.1(+) vector with the
CAG promoter. The TwinStrep-tagged P2 S was expressed in Expi293F cells.
Purification of the recombinant protein was based on a procedure described previously, withminor modifications (Cai et al, 2020). Upon cell lysis, P2 S was solubilised in 1% NP-40
detergent. The TwinStrep-tagged protein was then captured with StrepTactin Sepharose HP
resin in 0.5% NP-40. P2 S was further purified by size-exclusion chromatography and eluted
as three distinct peaks in 0.02 % NP-40 as previously reported (Cai et al, 2020). A peak thatconsists of intact P2 S migrating at around 150 kDa, as well as dissociated S1 and S2
subunits (which co-migrate at just above 75 kDa), was used in the structural characterization.
Spontaneous dissociation of the S1 and S2 subunits occurs throughout the course of protein
purification, starting at the point of detergent-mediated protein extraction, so that P2 S
preparations also contain dissociated S1 and S2.
3.3. Binding Kinetics of P2 S to Immobilized Human ACE2 and a Neutralizing
Monoclonal Antibody by Biolayer Interferometry
Binding of NP-40 solubilized, purified P2 S to ACE2-PD and human neutralizing
monoclonal antibody B38 (Wu et al, 2020) was measured by biolayer interferometry at 25 °C
on an Octet RED384 (FortéBio). P2 S binding was measured in 25 mM Tris pH 7.5, 150 mM
NaCl, 1 mM EDTA and 0.02% NP-40. Avi-tagged human ACE2-PD was immobilized on
streptavidin-coated sensors; B38 antibody was immobilized on protein G-coated sensors. For
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Vaccine Antigen
VR-VTR-10741, Ver. 2.0

a P2 S concentration series, after initial baseline equilibration of 120 seconds, the sensors
were dipped in a 10 µg/mL solution of Avi-tagged ACE2-PD or B38 mAb for 300 seconds toachieve capture levels of 1 nM using the threshold function. Then, after another 120 seconds
of baseline, binding data were collected for 300 seconds of association and 600 seconds ofdissociation.
Biolayer interferometry data were collected with Octet Data Acquisition software version
10.0.0.87 and processed using FortéBio Data Analysis software version 10.0. Data werereference subtracted and fit to a 1:1 binding model with R2 value greater than 0.95 todetermine kinetics and affinity of binding using Octet Data Analysis Software v10.0(FortéBio).

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3.4. Cryo-EM of P2 S
For TwinStrep-tagged P2 S, 4 μL purified protein at 0.5 mg/mL were applied to gold
Quantifoil R1.2/1.3 300 mesh grids freshly overlaid with graphene oxide. The sample wasblotted using a Vitrobot Mark IV for 4 seconds with a force of -2 before being plunged into
liquid ethane cooled by liquid nitrogen. 27,701 micrographs were collected from two
identically prepared grids. Data were collected from each grid over a defocus range of -1.2 to-3.4 μm with a total electron dose of 50.32 and 50.12 e-/Å2, respectively, fractionated into
40 frames over a 6-second exposure for 1.26 and 1.25 e-/Å2/frame. On-the-fly motion
correction, CTF estimation, and particle picking and extraction with a box size of 450 pixels
were performed in Warp (Tegunov & Cramer, 2019), during which super-resolution data
were binned to give a pixel size of 0.87 Å. A total of 1,119,906 particles were extracted. All
subsequent processing was performed in RELION 3.1-beta (Zivanov et al, 2018). Particle
heterogeneity was filtered out with 2D and 3D classification, yielding a set of 73,393
particles, which refined to 3.6 Å with C3 symmetry. 3D classification of this dataset without
particle alignment separated out one class with a single RBD up, representing 15,098
particles. The remaining 58,295 particles, in the three RBD ‘down’ conformation, wererefined to give a final model at 3.29 Å. The atomic model from PDB ID 6XR8(Cai et al, 2020) was rigid-body fitted into the map density, then flexibly fitted to the density
using real-space refinement in Phenix (Adams et al, 2010) alternating with manual buildingin Coot (Emsley et al, 2010). Data collection, 3D reconstruction and model refinement
statistics are listed in Table 1.

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Table 1.

CryoEM Data Collection, 3D Reconstruction and Refinement Statistics

Electron microscopy equipment
Voltage (keV)
Detector
Energy filter
Nominal magnification
Pixel size (Å)

090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)Electron dose (e-/Å2)
Dose rate (e-/Å2/sec)
Defocus range (m)
Number of collected micrographs
Number of selected micrographs
SoftwareNumber of used particles
Symmetry imposed
Global resolution (Å)
Fourier shell correction = 0.143
Applied B factor (Å2)

Data Collection
Titan Krios (Thermo Fisher Scientific)
300
K2 Summit
Gatan GIF, 20 ev slit
165,000 x
0.435 (super-resolution)
Grid 1
Grid 2
50.32
50.12
8.4
8.33
-1.2 to -3.4
-1.2 to -3.4
10,422
17,27927,701
3D Reconstruction
Warp, Relion
58,295C3
3.29-50
Refinement

SoftwareProtein residues
Map correlation coefficient
Root mean square deviation
Bond length (Å)
Bond angles ()Ramachandran plot statistics (%):
Preferred
AllowedOutlier
Poor rotamers (%)
MolProbity score
EMRinger score
Clashscore (all atoms)

Phenix, Coot
2,919
0.82
0.011
0.962
90.4
9.59
0
11.06
2.96
2.23
13.23

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4. RESULTS AND DISCUSSION
To confirm surface expression of untagged P2 S as well as the ability of P2 S to bind tohuman ACE2, flow cytometry experiments were performed on nonpermeabilized cells(Figure 1). Antibodies to the RBD, S1, and S2 were pre-incubated with Alexa-488 anti-IgG
Fab for staining, and a nucleic acid dye was used to separate live and dead cells. To confirm
binding of human ACE2, P2 S-expressing cells were labeled with the extracellular domain ofhuman ACE2 pre-incubated with a FITC-labeled antibody against an affinity tag on theACE2. Finally, anti-RBD human neutralizing antibodies B38 and H4 isolated from a
COVID-19 convalescent patient (Wu et al, 2020) and the anti-RBD therapeutic antibody
CR3022 (Yuan et al, 2020) were similarly confirmed to bind the surface-expressed P2 S.

090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)

Figure 1. Binding to Cell Surface-Expressed Recombinant P2 S

P2 S antigen was expressed in Expi293F cells, and surface expression confirmed by staining with antibodies
against the RBD, S1, and S2 regions of the full-length S protein. Human ACE2 peptidase domain as well as thetherapeutic antibody CR3022 and two neutralizing antibodies isolated from a COVID-19 convalescent patient,
B38 and H4, were further confirmed to bind to surface express P2 S. The nucleic acid dye 7-AAD was used
identify viable cells (lower quadrants in flow plots). Binding to surface expressed P2 S over background in live
cells is quantified across replicates in the bar graph.

For structural and biophysical characterization, P2 S was expressed in Expi293F cells from
DNA that encodes the same amino acid sequence as BNT162b2 RNA, with the addition of a
C-terminal TwinStrep tag for affinity purification. Following purification, as described in
Methods, P2 S eluted as three distinct peaks in 0.02% NP-40 as previously reported(Cai et al, 2020). Protein from the first peak of a size exclusion column, containing intact P2
S and dissociated S1 and S2, was assayed by biolayer interferometry (Figure 2). The trimeric
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P2 S bound to the human ACE2-PD, and an anti-RBD human neutralizing antibody B38 withhigh affinity (apparent KD = 1 nM).

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Figure 2. Biolayer Interferometry Sensorgrams for Binding of P2 S to ACE2-PD and
B38 mAbP2 S with a C-terminal TwinStrep tag expressed in Expi293F cells, was detergent solubilized and purified byaffinity and size exclusion chromatography. Protein from the first peak of a size exclusion column, containing
intact P2 S and dissociated S1 and S2, was assayed by biolayer interferometry on an Octet RED384 (FortéBio)at 25 °C in running buffer consisting of 25 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA and 0.02 % NP-40.
Sensorgrams showing the binding kinetics of TwinStrep-tagged P2 S to immobilized A, human ACE2-PD andB, B38 monoclonal antibody. The highest concentration tested for P2 S was 71 nM with 2 more 3-folddilutions. The binding curves were globally fit to a 1:1 Langmuir binding model with R2 values greater than
0.95. Actual binding data (black) and the best fit of the data to a 1:1 binding model (green). Apparent kinetic
parameters are provided in the graphs.

Purified TwinStrep-tagged P2 S was characterized structurally using cryoEM. 2Dclassification of particles from cryoEM data revealed a particle population that closely
resembles the prefusion conformation of SARS-CoV-2 spike protein (Figure 3A). Processing
and refinement of this dataset yielded a high-quality 3D map with a nominal resolution of
3.29 Å (Figure 3B), into which a previously published atomic model (PDB ID: 6VSB) was
fitted and rebuilt. The rebuilt model (Figure 3C) shows good agreement with reported
structures of prefusion full-length wild type S (Cai et al, 2020) and its ectodomain with P2
mutations (Wrapp et al, 2020). Three-dimensional classification of the dataset (Figure 3D)
showed a class of particles that was in the one RBD ‘up’ (accessible for receptor binding),
two RBD ‘down’ (closed) conformation and represented 20.4% of the trimeric molecules.
The remainder were in the all RBD ‘down’ conformation. The RBD in the ‘up’ conformation
was less well resolved than other parts of the structure, suggesting conformational flexibility
and a dynamic equilibrium between RBD ‘up’ and RBD ‘down’ states as also suggested byothers (Cai et al, 2020; Henderson et al, 2020).

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Figure 3. CryoEM P2 S Structure at 3.29 Å Resolution

A. Representative 2D class averages of TwinStrep-tagged P2 S particles extracted from cryoEM micrographs.
Box edge: 39.2 nm. B. Fourier shell correlation curve from RELION gold-standard refinement of the P2 S
trimer. C. 3.29 Å cryoEM map of TwinStrep-tagged P2 S, with fitted atomic model, showing top (perpendicular
to the three-fold axis) and side (parallel to the three-fold axis) views. CryoEM model is based on PDB 6VSB
and was fitted into the structure using manual rebuilding in Coot and real-space refinement in Phenix.
~28,000 micrographs were collected using a Titan Krios electron microscope operating at 300 kV accelerating
voltage, and image processing and 3D reconstructions were performed using Warp and RELION. D. Flowchart
for cryo-EM data processing of the complex, showing 3D class averages. Maps of P2 S produced by 3Dclassification indicate some heterogeneity in positioning of the RBD domains. Percentages of the particle
population represented in each class are indicated below the models.

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5. CONCLUSION
We demonstrate that the BNT162b2 RNA sequence encodes a recombinant P2 S that canauthentically present the ACE2 binding site and other epitopes targeted by SARS-CoV-2
neutralizing antibodies.
Binding of cell surface expressed P2 S to human ACE2 receptor and a panel of humanneutralizing mAbs was confirmed in cells using flow cytometry. Protein expressed from
DNA with the BNT162b2-encoded P2 S amino acid sequence was confirmed to be in theprefusion conformation by cryo-EM. This analysis showed that the antigenically important
RBD can assume the ‘up’ conformation, with the receptor binding site, rich in neutralizing
epitopes, accessible in a proportion of the molecules (Zost et al, 2020). The alternative statesobserved reflect a dynamic equilibrium between RBD ‘up’ and ‘down’ positions(Cai et al, 2020; Henderson et al, 2020). Binding of expressed and purified P2 S to ACE2 anda neutralizing monoclonal antibody further demonstrates its conformational and antigenic
integrity.
6. DEVIATIONS

090177e195e446ea\Approved\Approved On: 27-Dec-2020 02:23 (GMT)

N/A
7. REFERENCES
Adams PD, Afonine PV, Bunkoczi G, et al. PHENIX: a comprehensive Python-based system
for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010; 66:213-21.
Brouwer PJM, Caniels TG, van der Straten KJ, et al. Potent neutralizing antibodies fromCOVID-19 patients define multiple targets of vulnerability. Science 2020;369(6504):643-50.
Cai Y, Zhang J, Xiao T, et al. Distinct conformational states of SARS-CoV-2 spike protein.
Science 2020; 369:1586-92.
Emsley P, Lohkamp B, Scott WG, et al. Features and development of Coot. Acta Crystallogr
D Biol Crystallogr. 2010;66(Pt 4)(Apr):486-501.
Henderson R, Edwards RJ, Mansouri K, et al. Controlling the SARS-CoV-2 spikeglycoprotein conformation. Nat Struct Mol Biol 2020; 27:925-33.
Liu L, Wang P, Nair MS, et al. Potent neutralizing antibodies against multiple epitopes on
SARS-CoV-2 spike. Nature 2020;584(7821):450-6.
Pallesen J, Wang N, Corbett KS, et al. Immunogenicity and structures of a rationallydesigned prefusion MERS-CoV spike antigen. Proc Natl Acad Sci USA
2017;114(35)(08):E7348-E7357.
Robbiani DF, Gaebler C, Muecksch F, et al. Convergent antibody responses to SARS-CoV-2
in convalescent individuals. Nature 2020, 584, 437-42.

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Tegunov D, Cramer P. Real-time cryo-electron microscopy data preprocessing with Warp.
Nat Methods 2019; 16:1146–52.
Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in theprefusion conformation. Science 2020;367(6483)(03):1260-3.
Wu Y, Wang F, Shen C, et al. A noncompeting pair of human neutralizing antibodies block
COVID-19 virus binding to its receptor ACE2. Science 2020;368(6496)(06):1274-8.
Yuan M, Wu NC, Zhu X, et al. A highly conserved cryptic epitope in the receptor binding
domains of SARS-CoV-2 and SARS-CoV. Science 2020;368(6491)(05):630-3.
Zhou M, Zhang X, Qu J. Coronavirus disease 2019 (COVID-19): a clinical update. Front
Med 2020; 14:126‐135.
Zivanov J, Nakane T, Forsberg BO, et al. New tools for automated high-resolution cryo-EM
structure determination in RELION-3. eLife 2018;7:e42166.

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Zost SJ, Gilchuk P, Chen RE, et al. Rapid isolation and profiling of a diverse panel of humanmonoclonal antibodies targeting the SARS-CoV-2 spike protein. Nat Med 2020;26:1422-7.4.

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