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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 1
Title: Structural and Bioph ysical Characterization of SARS -CoV -2 Spike Gly coprotein
(P2S) as a Vaccine Antigen
Study Number: N/A
Parent Compound Number(s): PF-07302048
Alterna tive Compound Identifiers: N/A
Pfizer Discovery Sciences
Easte rnPoint Road
Groton , CT
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 2Title: Structural and Bioph ysical Characterization of SARS -CoV -2 Spike Gly coprotein
(P2S) as a Vaccine Antigen
PRINCIPAL INVESTIGATOR:
CONTRIBUTING SCIENTIST (S
):
PREPARED BY:
APPROVED BY:
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(b) (6)
(b) (6)
(b) (6)
(b) (6)
FDA-CBER-2021-5683-0709158
PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 3Title: Structural and Bioph ysicalCharacteriza tion of SARS -CoV -2 Spike Gly coprotein
(P2S) as a Vaccine Antigen
SYNOPSIS
The binding and structural anal ysis of SARS -CoV -2 P2 S expressed from DNA thatencodes
the same amino acid sequence asBNT162b2 R NAindicate that the en coded P2 S antigen
authentically present sthe ACE2 binding site and other epitop es targeted b y SARS -CoV -2
neutralizing antibodies.
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 4TABLE OF CONTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 4
LIST OF FIGURES ................................ ................................ ................................ ................... 4
1.OBJECTI VES ................................ ................................ ................................ ........................ 5
2.INTRODUCTION ................................ ................................ ................................ ................. 5
3.MATERIAL S AND METHODS ................................ ................................ ........................... 6
3.1.Flow Cy tometry Analy sis 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 I mmobilized Human ACE2 and a Neutralizing
Monoclonal Antibody by Biolay er Interferometry................................ ...................... 6
3.4.Cryo-EM of P2 S ................................ ................................ ................................ .......7
4.RESUL TS AND DI SCUSSION ................................ ................................ ............................ 9
5. CONCL USION ................................ ................................ ................................ .................... 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. Biolay er Interferometry Sensorgrams for Binding of P2 S to ACE2-
PD and B38 mAb ................................ ................................ ...................... 10
Figure 3. CryoEM P2 S Structure at 3.29 Å Resoluti on ................................ .......... 11
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 5Title: Structural and Bioph ysical Characterization of SARS -CoV -2 Spike Gly coprotei n
(P2S) as a Vaccine Antigen
Study Number: N/A
Functional Area: Medicinal Sciences
Test Facility :Pfizer Discovery Sciences, Eastern Point Roa d,
Groton, CT
Study/Testin gInitiation Date: 07April2020
Study/Testing Completi on Date: 19Aug2020
1.OBJECTIVES
The pur pose of this study was to express and characterize the vaccine antig enencoded b y
BNT162b2 .
2.INTRODUCTION
The coronavirus disease 2019 (COV ID-19) vaccine (BioNTech code number BNT162, Pfizer
code number PF -07302048) is aninvestigational vaccine intended toprevent COVID -19,
which is caused bysevere acute respiratory syndrome coronavirus 2 (SARS -CoV -2).
Coronavirus S is a major target o f virus neutralizing antibodie s and is a key antigen for
vaccine development. S is a transmembrane gl ycoprotein responsible for receptor
recognition, attachment to the cell, and viral envelop e fusion with a host cel l membrane
resulting in genome release . While the membrane -proximal S2 is responsible for membrane
fusion, the membrane -distal S1, with its receptor- binding domain (RBD), recognizes the host
receptor, angiotensin converting enzy me 2 (ACE2) (Zhou et al, 2020). The RBD forms
membrane distal “heads” on the S trimer that are conn ected to the bod y by ahinge. In th e
native S, the RBD alternates between an open (up) and closed (down) position. Although
potent neutralizing epitopes have bee n described when the RBD is in the “heads down”
closed conformati on, the “heads up” receptor ac cessible conformation exposes a potentially
greater breadth of neutralizing antibody targets ( Brouwer et al, 2020 ; Liu et al, 2020 ;
Robbiani et al, 2020 ).
The gl ycoprotein encoded by the vaccine candidate BNT162 b2 includes two amino acid
substitutions to proline (P2 S) locking the transmemb rane protein in an antigenically optimal
prefusion conformation ( Pallesen et al, 2017 ; Wrapp et al, 2020 ).The P2 S antigen wa s
expressed from DNA and characterized for structure and binding to hu man ACE2 and
SAR S-CoV -2 neutralizing antibodies .
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 63.MATERIALS AND METHODS
3.1.Flow Cytometry Analysis of Binding to Cell Surface -Expressed P2 S
Amodified pcDN A3.1 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 .
Cells were collected 48 hr post transfection, washed with TBS buffer, and use d at 4- 5 x 104
for each condition. Cells were incubated fo r 1 hrat room tempe rature ( RT)inTBS + 4%
BSA + 0.01 mg/mL 7-AAD to detect non -viable cells and with either (i) 1:100 FITC -labeled
anti-6xHis plus 10 nM His -tagged human ACE2 peptidase domain (ACE2 -PD); (ii) 100 nM
Alexa-488 labeled anti -Rabbit IgG Fab plus eit her 3 3 nM anti -SARS -CoV -2 Spik e 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 thera peutic 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 anal yzedin a
V-bottom 96 -well plate using a Guava Eas yCyte HT flow cy tomet ry system.For each
condition, 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 f or biophy sical characteri zation, a
gene encoding the full length SARS -CoV -2spike (GenBank: MN908947) with two proli nes
substituted at residues 986 and 987 (K986P and V987P) followed with a C-terminal HRV3 C
protease site and a TwinStre p tag was cloned into a modified pcDNA3.1(+) vector with the
CAG promoter. The TwinStrep -tagged P2 S was expressed in Expi293 Fcells.
Purification of the recombinant protein was based on a procedure described previously , with
minor modifications ( Caiet al, 2020 ). Upon cell lysis, P2 S was solubilised in 1% NP -40
detergent. The TwinStrep- tagged protein was then captured with StrepTactin Sep harose HP
resin in 0.5% NP -40. P2 S was further purified b y size-exclusion chromatograph yand eluted
as three distinct peaks in 0.02 % NP- 40 as previously reported (Caiet al, 2020). A peak that
consists of int act P2 S migrating at around 150 kDa, as well as dissociated S1 and S2
subunits (which co -migrate at just ab ove 75 kDa),was used in the str uctural characteri zation.
Spontaneous dissociation of the S1 and S2 subunits occurs throughout the course of protein
purification, starting at the point of deterg ent-mediated protein extraction, so that P2 S
preparations also cont ain d issoc iated S1 and S2 .
3.3.Binding Kinetics of P2 S to Immobilized Human ACE2 and a Neutralizing
Monoclonal Antibody by Biolayer Interferomet ry
Binding of NP-40 solubili zed, purified P2 S to ACE2 -PD and human neutrali zing
monoclonal antibody B38 (Wu et al, 2020) wasmeasured b y biolay er 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 hu man ACE2- PD was immobili zed on
streptavidin -coated sensors; B38 an tibody was immobili zed on protein G -coated sensors. For
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 7a P2 S concentration series, after initial baseline equilibration of 120 s econds , the sensors
were dipped in a 10 µg/mL solution of Avi- tagged ACE2 -PD or B3 8 mAb for 300 s econds to
achieve captur e lev els of 1 nM using the thr eshold function. Then, after another 120 seconds
of baseline, binding data were collected for 300 seconds o f association and 600 seconds of
dissociation.
Biolay er interferometry data were collected with Octet Dat a Acquisition software version
10.0.0.87 and processed using FortéBio Data Analy sis software version 10.0. Data were
reference subtracted and fit to a 1:1 binding model with R2value greater than 0. 95 to
determine kinetics and affinity of binding using Oct et Data Anal ysis Software v10.0
(FortéBio).
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
Quanti foil R1.2/1.3 300 mesh grids freshl y overlaid wit h graphene oxide. The s ample was
blotted using a Vitrobot Mark IV for 4 s econds with a force of - 2 before being plunge d into
liquid ethane cooled b y liquid nitrogen. 27,701 micrographs were collected from tw o
identically prepared grids. Data were collected from each grid over a defocus range of - 1.2 to
-3.4μm with a total electron dose of 50.32 and 50.12 e-/Å2, respectivel y, fractionated into
40frames 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, y ielding a set of 73,393
particles, which refined to 3.6 Å with C3 sy mme try. 3D classification of this dataset without
particle alignment separated out on e class with a single RBD up, representing 15,098
particles. The remaining 58,295 par ticles, in the three RBD ‘down’ conformation, were
refined to give a final model at 3.29 Å. The atomic model from PDB ID 6XR8
(Caiet al, 2020 )was rigid -body fitted i nto the map d ensity , then flexibly fitted to the density
using real -space refinement in Phenix (Adam set al, 2010) alternating with manual building
in Coot (Emsley et al, 2010 ). Data collection, 3D reconstruction an d model refinement
statistics are l isted in Table 1.
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Vaccine Antigen
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PFIZER CONFIDENT IAL
Page 8Table 1. C ryoEM Data Collection, 3D Reconstruction and Refinement Statistics
Data C ollection
Electron micro scopy equipment Titan Krios (Thermo Fisher Scientific)
Voltage (keV) 300
Detector K2 Summit
Energy filter Gatan GIF, 20 ev s lit
Nom inal magnification 165,000 x
Pixel size (Å) 0.435 (super -resolution)
Grid 1 Grid 2
Electron dose (e-/Å2) 50.32 50.12
Dose rate (e-/Å2/sec) 8.4 8.33
Defocus range ( m) -1.2to -3.4 -1.2 to - 3.4
Number of collected micrographs 10,422 17,279
Number of selected mic rographs 27,701
3D Reconstruction
Software Warp, Relion
Number of used particles 58,295
Symmetry imposed C3
Global resolution ( Å)
Fourier shell correction = 0.143 3.29
Applied B factor ( Å2) -50
Refinement
Software Phenix, Coo t
Prote in residues 2,919
Map correlation coefficient 0.82
Root mean square deviation
Bond length ( Å) 0.011
Bond angles ( ) 0.962
Ramachandran plot statistics (%):
Preferred 90.4
Allowed 9.59
Outlier 0
Poor rot amers (%) 11.06
MolProbity score 2.96
EMR inger score 2.23
Clashscore (all atoms) 13.23
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Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
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Page 94.RESULTS AND DISCUSSION
To confirm surface expre ssion of untagged P2 S as well as the ability of P2 S to bind to
human ACE2, flow cytometry experiments were perfor med 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 dy e was used to separate live and dead ce lls. To confirm
binding of human ACE2, P2 S- expressing cells were labeled with the extracellular domain of
human ACE2 pre -incubated with a FITC- labeled antibody against an aff inity tag on the
ACE2. Finally , anti- RBD human neutralizing antibodies B38 and H4 isolated from a
COVID -19 convalescent patient ( Wu et al, 2020) andthe anti- RBD therapeutic antibody
CR3022 ( Yuan et al, 2020 ) were similarly confirmed to bind the surface -expressed P2 S.
Figure 1.Binding to Cell Surface -Expresse d Recombinant P2 S
P2 S antigen was expressed in Expi293F cel ls, and surface express ion 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 the
therapeutic antibod y CR3022 and tw o neutralizin g 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 t o surface expressed P2 S over background in live
cells is quant ified across replicates in the bar g raph.
For structural and bioph ysical 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 affinit y purification .Followi ng purificati on,as described in
Methods, P2 S elute d as three distinct peaks in 0.02% NP -40 as previousl y reported
(Caietal,2020 ). Protein from the first peak of a si ze exclu sion column, containing intact P2
S and dissociated S1 and S2, was assay ed by biolayer interferometry ( Figure 2). The trimeric
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 10P2 S bou nd to the human A CE2-PD, and an anti -RBD human neutralizing antibody B38 with
high affinity (apparent KD= 1nM).
Figure 2.Biolay er Interfe rometry Sensorgrams for Binding of P2 S to ACE2 -PD and
B38 mAb
P2 S w ith a C -terminal T winStrep tag exp ressed in Expi293F cells, was deterge nt solubilized and purified by
affinity and size exclusion chromatography. Protein from the first peak o f a size exclusion column, containing
intact P2 S and dissociated S1 and S2, was assayed by biolayer interferometry on an Octet RED384 (Forté Bio)
at 25 ° C in running buffer consisting of 25 mM Tris p H 7.5, 150 mM NaCl, 1mM EDTA and 0.02 % N P-40.
Sensorgram s showing the binding kinetics of T winStrep -tagged P2 S to immobilized A,human ACE2 -PD and
B,B38 monoclonal antib ody. The highest con centra tion tested for P2 S w as 71 nM with 2 more 3 -fold
dilution s. The binding curves were globally fit t o a 1:1 Langmuir binding model with R2values 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 Twin Strep-tagged P2 S was characterized structurally using cry oEM. 2D
classification of part icles from cry oEM data rev ealed a particle population that closely
resembles the prefusion conformation of SARS -CoV -2 spike protein (Figure 3A). Processing
and refinement of this dataset y ielded a high- quality 3D ma p with a nominal resolution of
3.29 Å ( Figure 3B), into which a previousl y published atomi c model (PDB ID: 6VS B) was
fitted and rebuilt. The rebuilt model (Figure 3C) shows good agr eement with reported
structures of prefusion full -length wild t ype S (Caiet al, 2020) and its ectodomain with P2
mutations ( Wrapp et al, 2020 ). Three -dimensional classification of the data set (Figure 3D)
showed a class of particle s that was in the one RBD ‘up’ (accessible for receptor binding),
two RBD ‘down ’(closed) conformation and rep resented 20.4% of the trimeric molecules.
The remainder were in the all R BD ‘down’ conformation. The RBD in the ‘up’ conformation
was less well re solved tha n other parts of the structu re, suggesting conformational flexibility
and a d ynamic equilibrium betw een RBD ‘up’ and RBD ‘down’ states as also suggested b y
others ( Caiet al, 2020; Henderson et al, 2020).
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Vaccine Antigen
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Page 11Figure 3.CryoEM P2 S Structure at 3.29 Å Resolution
A. Representative 2D class averages of T winStrep -tagged P2 S parti cles extracted from cryoEM micrographs.
Box edge: 39.2 nm. B.Four ier shell correlation curve from RELION gold -standard refinement of the P2 S
trimer.C. 3.29 Å cryoEM map of Tw inStrep -tagged P2 S, with fitte d atom ic model, showing top (perpendicular
to the three -fold axis) and side (parallel to the three -fold axis) view s. CryoEM model is based on PDB 6VSB
and w as fitted into the structure using manu al rebuilding in Coot and real -space re finement in Phenix.
~28,000 micrographs were collected using a Titan Krios electron microscope operating at 300 kV accelerating
voltage, and im age processing and 3D reconstructions were performed using Warp and RELION . D.Flowchart
for cryo -EM data process ing of the complex, sh owing 3D class averages. Maps of P2 S produced b y 3D
classification indicate some heterogeneity in positioning of the RBD domains. Percentages of the particle
population represented in each class areindicated below the models.
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PF-07302048 : Structural and Biophysical Char acterization of SARS -CoV -2 Spike Glycoprotein (P2 S) as a
Vaccine Antigen
VR-VTR -10741 , Ver. 2.0
PFIZER CONFIDENT IAL
Page 125.CONC LUSION
We demonstrate that the BNT162b2 RNA sequence encodes a recombin ant P2 S that can
authentically present the ACE2 binding site and other epitopes targeted by SARS -CoV -2
neutralizing antibodies.
Binding of cell s urfac e expressed P2 S to human ACE2 rec eptor and a panel of h uman
neutralizing mAbs was confirmed in cells usi ng flow cy tometry . Protein expressed from
DNA with the BNT162b2- encoded P2 S amino acid sequence was confirmed to be in the
prefusion conformation by cryo-EM. This analy sis 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 molec ules(Zost et al, 2020 ). The alternative states
observ ed refl ect a dy namic equilibrium between RBD ‘up’ and ‘down’ p ositio ns
(Caietal,2020 ; Henderson et al, 2020) . Binding of expressed and purified P2 S to ACE2 and
a neutralizing monoclonal antibody further demonstrates its conformational and ant igeni c
integrity.
6.DEVIATIONS
N/A
7.REFERENCES
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Robb iani D F, 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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Page 13Tegunov D,Cramer P. R eal-time cry o-electron microscop y data preprocessing with Warp.
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Yuan M, Wu NC, Zhu X, et al. A highl y conserved cry ptic epitope in the receptor binding
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Zhou M, Zha ng X, Qu J. Coronavirus d isease 2019 (COVID -19): a clinical update. Front
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Zivanov J , Nakane T, Forsberg BO, et al. New tools for automated high- resolution cry o-EM
structure determinatio n in RELION -3. eLife 2018;7:e42166.
Zost SJ, Gilchuk P, Chen RE ,et al. Rapid isol ation and profiling of a diverse panel of human
monoclon al antibodies targeting the SARS -CoV -2 spike protein. Nat Med 2020; 26:1422 -7.4.
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