125742 S1 M2 24 nonclinical overview

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BNT162b2
Module 2.4. Nonclinical Overview
2.4 NONCLINICAL OVERVIEW
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TABLE OF CONTENTS
2.4 NONCLINICAL OVERVIEW ..........................................................................................
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ...................................................
2.4.1. OVERVIEW OF NONCLINICAL TESTING STRATEGY ...............................................
Table 2.4.1-1.  Nomenclature of the Vaccine Candidates ........................................................Table 2.4.1-2.  Nonclinical Studies ..........................................................................................
2.4.2. PHARMACOLOGY .............................................................................................................
2.4.2.1. Primary Pharmacodynamics ........................................................................................
2.4.2.1.1. Summary ............................................................................................................2.4.2.1.2. BNT162b2, A Lipid Nanoparticle Encapsulated RNA Vaccine Encoding the
SARS-CoV-2 P2 S as a Vaccine Antigen...........................................................................
Figure 2.4.2-1. Schematic of the Organization of the SARS-CoV-2 S Glycoprotein .......
2.4.2.1.3. Immunogenicity of BNT162b2 (V9) in Mice ....................................................
2.4.2.1.4. Evaluation of BNT162b2 (V9) Immunogenicity and Protection Against 
SARS-CoV-2 Challenge in Rhesus Macaques...................................................................
2.4.2.1.5. Immunogenicity Testing After Weekly Immunization of Rats in GLP
Compliant Repeat Dose Toxicology Studies and a Developmental and ReproductiveToxicity Study...................................................................................................................
2.4.2.2. Secondary Pharmacodynamics ..................................................................................
2.4.2.3. Safety Pharmacology .................................................................................................
2.4.2.4. Pharmacodynamic Drug Interactions ........................................................................
2.4.3. PHARMACOKINETICS ....................................................................................................
2.4.3.1. Brief Summary ..........................................................................................................
2.4.3.2. Methods of Analysis ..................................................................................................
2.4.3.3. Absorption .................................................................................................................
2.4.3.3.1. In Vitro Absorption ..........................................................................................2.4.3.3.2. Single-Dose Pharmacokinetics ........................................................................
Table 2.4.3-1.  PK of ALC-0315 and ALC-0159 in Wistar Han Rats After IV
Administration of LNPs Containing Surrogate Luciferase RNA at 1 mg/kg..........................
Figure 2.4.3-1. Plasma and Liver Concentrations of ALC-0315 and ALC-0159 in Wistar
Han Rats After IV Administration of LNPs Containing Surrogate Luciferase RNA at 1mg/kg.......................................................................................................................................
2.4.3.4. Distribution ................................................................................................................
Figure 2.4.3-2. Bioluminescence Emission in BALB/c Mice after IM Injection of an LNP
Formulation of modRNA Encoding Luciferase......................................................................1
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2.4.3.5. Metabolism ................................................................................................................
Figure 2.4.3-3. Proposed Biotransformation Pathway of ALC-0315 in Various Species ......
Figure 2.4.3-4.  Proposed Biotransformation Pathway of ALC-0159 in Various Species .....
2.4.3.6. Excretion ...................................................................................................................
2.4.3.7. Pharmacokinetic Drug Interactions ...........................................................................
2.4.4. TOXICOLOGY ..................................................................................................................
2.4.4.1. Brief Summary ..........................................................................................................
Table 2.4.4-1.  Overview of Toxicity Testing Program .........................................................
2.4.4.2. Single-Dose Toxicity .................................................................................................
2.4.4.3. Repeat-Dose Toxicity ................................................................................................
2.4.4.3.1. Repeat-Dose Toxicity Study of Three LNP-Formulated RNA Platforms
Encoding for Viral Proteins by Repeated Intramuscular Administration to Wistar HanRats...................................................................................................................................
2.4.4.3.2. 17-Day Intramuscular Toxicity Study of BNT162b2 (V9) in Wistar Han
Rats with a 3-week Recovery...........................................................................................
2.4.4.4. Genotoxicity ..............................................................................................................
2.4.4.5. Carcinogenicity .........................................................................................................
2.4.4.6. Reproductive and Developmental Toxicity ...............................................................
2.4.4.7. Local Tolerance .........................................................................................................
2.4.4.8. Other Toxicity Studies ...............................................................................................
2.4.4.8.1. Phototoxicity ....................................................................................................2.4.4.8.2. Antigenicity ......................................................................................................
2.4.4.8.3. Immunotoxicity ................................................................................................
2.4.4.8.4. Mechanistic Studies .........................................................................................
2.4.4.8.5. Dependence ......................................................................................................
2.4.4.8.6. Studies on Metabolites .....................................................................................2.4.4.8.7. Studies on Impurities .......................................................................................
2.4.4.8.8. Other Studies ....................................................................................................
2.4.4.9. Target Organ Toxicity ...............................................................................................
2.4.5. INTEGRATED OVERVIEW AND CONCLUSIONS ......................................................
2.4.6. LIST OF LITERATURE REFERENCES ..........................................................................1819
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LIST OF ABBREVIATIONS AND DEFINITION OF T ERMS
A:G Albumin:globulin ratio
ACE Angiotension -converting enzy me
ADME Absorption, distribution, metabolism, excretion
ALC-0159 Proprietary  PEG -lipid included as an excipient in the L NP formulation 
used in BNT162b2
ALC-0315 P roprietary  amino- lipid included as an excipient in the L NP formulation 
used in BNT162b2
ALT Alanine aminotransferase
AST Aspartate aminotransferase
BAL Bronchoalveolar lavage
CAS Chemical abstracts service
CBER Center for Biologics Evaluation and Research
CD Cluster of differentiation
COVID -19 Coronavirus Disease 2019
DART Developmental and reproductive toxicity
DNA Deox yribonucleic acid
DSPC 1,2-distearo yl-sn-glycero-3-phosphocholine
ELISA Enzy me-linked immunosorbent assay
EUA Emergency  Use Authorization
F0 Parental generation administered vaccine
F1 First generation offspring of F0 generation
GD Gestation day
GGT Gamma -glutam yl transferase
GLP Good Laboratory  Practice
H Human (in metabolite scheme)
[3H]-CHE Radiolabeled [Cholesteryl -1,2-3H(N)] -Cholestery l Hexadecy l Ether
HGB Hemoglobin
IFN Interferon
IgG Immunoglobulin G
IL Interleukin
IM Intramuscular(ly )
IND Investigational New Drug Application
IV Intravenous(l y)
LC/MS Liquid chromatograph y-tandem mass spectrometry
LD Lactation day
LNP Lipid -nanoparticle
Luc Luciferase (from firefl y Pyractomena lucifera )
LUC Large unstained cell
Mk Monkey  (in metabolite scheme)
Mo Mouse (in metabolite scheme)
modRNA Nucleoside -modified mRNA
mRNA Messenger RNA
NA Not applicable
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LIST OF ABBREVIATIONS AND DEFINITION OF T ERMS - CONTINUED
NHP Nonhuman primate
OECD Organisation for Economic Co -operation and Development
P2 S Spike protein P2 mutant
PEG Polyethylene gl ycol
PK Pharmacokinetics
PLT Platelet
PND Postnatal day
PT Prothrombin time
QC Quality  control review
QW Once weekl y
R Rat (in metabolite scheme)
RBC Red blood cell
RBD Receptor binding domain
RdRp RNA -dependent RNA -polymerase
RDW Red cell distribution width
RETI C Reticulocy te
RNA Ribonucleic acid
RT-PCR Reverse transcription -polymerase chain reaction
S SARS -CoV -2 spike gl ycoprotein
S1 S1 domain of the SARS -CoV -2 spike gl ycoprotein
S9 Supernatant fraction obtained from liver homogenate b y centrifuging at 
9000 g
SARS Severe Acute Respiratory S yndrome
SARS -CoV -2 Severe acute respiratory  syndrome coronavirus 2; coronavirus causing 
COVID -19
Tfh T follicular helper cell
Th1 Type 1 T helper cells
TK Toxicokinetic
TNF Tumor necrosis factor
V8 Variant 8; P2 S
V9 Variant 9; P2 S
WBC White blood cell
WHO World Health Organization
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Module 2.4. Nonclinical Overview
2.4.1. OVERVIEW OF NONCLINI CAL TESTING STRATEGY
BNT162b2 (BioNTech code number BNT162, Pfizer code number PF -07302048) is an
investigational vaccine intended toprevent COVID -19, which is caused bySARS -CoV -2.
BNT162b2 is a nucleoside modified mRNA (modRNA) expressing full -length S with two 
proline mutations (P2) to lock the transmembrane protein in an antigenica lly optimal 
prefusion conformation ( Pallesen et al, 2017 ; Wrapp et al, 2020 ). The vaccine is formulated 
in lipid nanoparticles (LNPs). The LNP is composed of 4 lipids: AL C-0315, AL C-0159, 
DSPC , and cholesterol. Other excipients in the formulation include sucrose, NaCl, KCl, 
Na2HPO 4, and KH 2PO 4. The dose selected for BNT162b2, with efficacy  demonstrated in
Phase 2/3 clinical evaluation and intended for commercial use, is 30 µg administered I M as
two doses given 21 day s apart.
In nonclinical studies, two variants of BNT162b2 were tested; designated “variant 8” and 
“variant 9” (V8 and V9, respectivel y). The variants differ onl y in their codon optimization 
sequences which are designed to improve an tigen expression, otherwise the amino acid 
sequences of the encoded antigens are identical. Only BNT162b2 (V9) has been evaluated in 
the clinic , is currentl y authorized under EUA, and is the subject of this BLA application. The 
characteristics of each vari ant are described in the table below (Table 2.4.1- 1).  
Table 2.4.1-1.  Nomenclature of the Vaccine Candidates
Product
Code RNA
PlatformAntigen
VariantDescription/ Translated Protein Variant
CodeGLP 
Tox 
DataClinical 
Candidate
BNT162b2 modRNA V8aP2S RBP020.1 Yes No
BNT162b2 modRNA V9aP2 S RBP020.2 Yes Yes
a.The V8 and V9 variants of the P2 S antigen have the same amino acid sequence. Different codon 
optimizations were used for their ribonucleotide sequences.
Bold : BNT162b2 (V9) vaccine candidate submitted for licensure.
The primary  pharmacology , distribution, metabolism, and safet y of BNT162b2 were 
evaluated in nonclinical pharmacology , pharmacokinetic, and toxicity  studies invitro and 
invivo (Table 2.4.1 -2).
Immunogenicit y of BNT162b2 was evaluated in mice ( 2.4.2.1.3 ), rats ( 2.4.2.1.5 )and 
nonhuman primates ( 2.4.2.1.4 ). For assessment of serum antibody  responses in mice and rats, 
S1 and RBD -binding IgG responses were tested by  an E LISA. Functional antibody responses 
were tested b y a SARS -CoV -2 pseudot ype neutralization assay (pVNT). In nonhuman 
primate studies, S1 -binding IgG responses were tested in a direct Luminex- based 
immunoassay (dLIA) a nd functional antibody  responses were assessed in a SARS -CoV -2 
neutralization assay . S-specific T cell responses were assessed in mouse and nonhuman 
primate studies in an IFN ELISpot and by  intracellular cy tokine staining flow cy tometry -
based analysis of the Th1/Th2 profile using splenocy tes.  
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A SARS -CoV -2 challenge study  in BNT162b2 (V9) -immunized nonhuman primates was 
also conducted to assess protection against infection and to demonstrate lack of disease 
enhancement (Section 2.4.2.1.4.2 ).
Platform properties that support BNT162b2 were initially  demonstrated with 
non-SARS -CoV -2 antigens. Non- GLP in vivo testing of an LNP -formulated modRNA 
encoding luciferase examined biodistribution in BALB/c mice and Wistar Han rats after IM 
injection ( Section2.4.3.4 ) and the PK of the two novel excipients in the L NP formulation, 
ALC-0315 and AL C-0159, in Wistar Han rats (Section 2.4.3.3 ). In addition, the metabolism 
of AL C-0315 and ALC -0159 was evaluated in mouse, rat, monkey , and human blood, liver 
micros omes, S9 fractions, and hepatocy tes and in vivo in rat plasma, urine, feces, and liver 
samples from the PK study  (Table 2.4.1 -2; Section 2.4.3.5 ).  
BNT162b2 (V8) and (V9) have been studied in GLP -compliant repeat -dose toxicity  studies 
in rats ( Table 2.4.1 -2). Two GL P repeat -dose toxicity  studies for BNT162b2 (V8) and 
BNT162b2 (V9), one study for each variant, have been completed. The study  designs are 
described in Section 2.4.4 and are based on WHO guidelines for vaccine development 
(WHO, 2005). ADART study  with BNT162b2 (V9) in rats has also been completed. No 
additional toxicity  studies are planned for BNT162b2.
IM administration was chosen for the toxicity  studies as this is the intended route of 
administration. Rats were chosen for toxicity  assessments as they  are a commonly  used 
animal species for the evaluation of toxicity , and they  mount an antigen -specific immune 
response to vaccination with BNT162b2 .
The design of the nonclinical rep eat-dose toxicity  studies was consistent with the WHO 
Guidelines on Nonclinical Evaluation of Vaccines, the EMA Note for Guidance on 
Preclinical Pharmacological and Toxicological Testing of Vaccines, and Japan guidance on 
the nonclinical safety  assessment of vaccines. In addition, the 2020 CBER guidance on 
“Development and L icensure of Vaccines to Prevent COVI D-19” ( US FDA, 2020 ) was 
considered when assembling the nonclinical safet y licensure package as well as feedback 
from regulatory  agencies. All GL P-compliant studies were conducted in accordance with 
Good Laboratory  Practice for Nonclinical Laboratory  Studies, Code of US Federal 
Regulations (21 CFR Part 58), in an OECD Mutual Acceptance of Data member state. All 
nonclinical studies described herein were conducted by  or for Pfizer Inc or BioNTech RNA 
Pharmaceuticals Gm bH. The location of records for inspection is included in each final study  
report.
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Table 2.4.1-2.  Nonclinical Studies
Study Number Study Type Species / 
Test 
SystemTest Item Dose 
[RNA]Cross reference
Pharm acology -BNT162b2 studies
R-20-0085 Invivo 
immunogenicityBALB/c 
miceBNT162b2 (V9) 0.2, 1, 
5µgSection 2.4.2.1.3
R-20-0112 Invivo 
immunogenicityBALB/c 
miceBNT162a1, 
BNT162b1, 
BNT162b2 (V9), 
BNT162c25 µg Section 2.4.2.1.3
R-20-0211 In vitro protein 
expressionCell culture BNT162b2 (V9) varied Section 2.4.2.1.2
VR-VTR -10741 In vitro protein 
expressionCell culture BNT162b2 (V9) varied Section 2.4.2.1.2
VR-VTR -10671 In vivo 
immunogenicity 
and 
SARS -CoV- 2 
challengeRhesus 
macaquesBNT162b2 (V9) 30 and 
100 µgSection 2.4.2.1.4
ADME
PF-07302048
_06Jul20_072424PK of 
ALC -0315 and 
ALC -0159Wistar Han 
RatsmodRNA encoding 
luciferase 
formulated in LNP 
comparable to 
BNT162b21 mg/kg Section 2.4.3.3
R-20-0072 Invivo 
distributionBALB/c
micemodRNA encoding 
luciferase 
formulated in LNP 
comparable to 
BNT162b22 µg Section 2.4.3.4
185350 In vivo 
distribution Wistar Han 
RatsmodRNA encoding 
luciferase 
formulated in LNP 
comparable to 
BNT162b2 w ith 
trace amounts of 
[3H]-CHE as non -
diffusible label50 µg Section 2.4.3.4
01049 -20008 In vitro
metabolismCD-1/ICR 
mouse, 
Wistar Han 
and/or 
Sprague 
Dawley rat, 
cynomolgus 
monkey and 
human liver 
microsomes, 
S9 fraction, 
hepatocytesALC -0315 NA Section 2.4.3.5
01049 -20009
01049 -20010
01049 -20020 ALC -0159 NA
01049 -20021
01049 -20022
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Table 2.4.1-2.  Nonclinical Studies - Continued
Study Number Study Type Species / 
Test 
SystemTest Item Dose 
[RNA]Cross reference
PF-07302048
_05Aug20_043725In vitro and in 
vivo 
metabolismBlood, liver 
S9 fractions 
and 
hepatocytes 
from CD-1 
mouse, 
Wistar Han
rat, 
cynomolgus 
monkey and 
human. In 
vivo 
samples 
from Wistar 
Han rat 
plasma, 
urine, feces, 
and liverIn vitro: ALC -0315 
and ALC -0159
In vivo: modRNA 
encoding luciferase 
formulated in LNP 
comparable to 
BNT162b21 mg/kg 
modRNA 
(in vivo 
samples)Section 2.4.3.5
Toxicology –Studies with BNT162b2 variants
38166 Repeat -dose 
toxicityWistar Han 
RatsBNT162b2 (V8) 100 µg Section 2.4.4.3
20GR142 Repeat -dose 
toxicityWistar Han 
RatsBNT162b2 (V9) 30 µg Section 2.4.4.3
20256434 Development 
and 
Reproductive 
ToxicityWistar Han 
RatsBNT162b2 (V9) 30 µg Section 2.4.4.6
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2.4.2.PHARMACOLOGY
2.4.2.1.Primary Pharmacodynamics
2.4.2.1.1. Summary
BNT162b2 (BioNTech code number BNT162, Pfizer code number PF -07302048) is a 
nucleoside -modified mRNA (modRNA) vaccine that encodes the SARS -CoV -2 full -length 
spike gl ycoprotein (S). The gl ycoprotein encoded by  both BNT162b2 variants includes two 
amino acid substitutions to proline (P2 S) locking the transmembrane protein in an 
antigenically  optimal prefusion conformation ( Wrapp et al, 2020 ; Pallesen etal,2017 ).The 
RNA is formulated with functional and structural lipids, which protect the RNA from 
degradation and enable transfection of the RNA into host cells after IM injec tion. S is a 
major target of virus neutralizing antibodies and is a key  antigen for vaccine development. 
The well -resolved trimeric prefusion structure and the high affinity  binding to ACE2 and 
human neutralizing antibodies demonstrate that the recombinant P2 S authentically  presents 
the ACE2 binding site and other epitopes targeted by  man y SARS -CoV -2 neutralizing 
antibodies.
In vitro studies and in vivo studies in mice and nonhuman primates demonstrate the 
mechanism of action for this RNA -based vaccine, which is to encode SARS -CoV -2 S that 
induces an immune response characterized by  both a strong neutralizing antibody  response 
and Th1- type CD4+and an IFN +CD8+T-cell response. BNT162b2 immunization protected 
rhesus macaques from infectious SARS- CoV -2 chal lenge, with reduced detection of viral 
RNA in vaccine -immunized animals compared to saline -immunized animals and with no 
evidence of clinical exacerbation.
2.4.2.1.2. BNT162b2, A Lipid Nanoparticle Encapsulated RNA Vaccine Encoding the 
SARS-CoV-2 P2 S as a Vaccine An tigen
BNT162b2 is based on a nucleoside- modified mRNA (modRNA) platform technology . 
Vaccination with modRNA formulated in LNPs is characterized b y strong expansion of 
Th1- skewed antigen -specific T follicular helper (Tfh) cells, which stimulate and expand 
germinal center B cells, thereb y resulting in particularly  strong, long lived, high- affinity  
antibody  responses ( Sahin et al, 2014; Pardi et al, 2018 ). ModRNA vaccine candidates 
against other infectious diseases induce strong antibody  responses and prime an d expand 
multifunctional CD4+and CD8+T cells ( Pardi et al, 2017 ; Pardi et al, 2018).
SARS -CoV -2 S is a large, trimeric gly coprotein that exists predominantly  in a prefusion 
conformation on the virion ( Ke et al, 2020 ). It is cleaved b y furin into an N-terminal S1 and a 
C-terminal S2 fragment. S attaches to the host cell receptor, ACE2, by its receptor binding 
domain which is contained in the S1 furin cleavage fragment. Spontaneously  and during cell 
entry , the S1 fragment dissociates, and the S2 fragme nt undergoes a fold -back rearrangement 
to the post -fusion conformation in a process that facilitates fusion of viral and host cell 
membranes. S is the main target of virus neutralizing antibodies (Zakhartchouk et al,2007 ; 
Yong et al, 2019 ).Most of the antibodies with SARS -CoV -2 neutralizing activity  are 
directed against the RBD ( Jiang et al, 2020 ; Zost et al, 2020 ).
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Figure 2.4.2 -1.Schematic of the Organization of the SARS -CoV-2 S Glycoprotein
The S1 furin cleavage fragment includ es the signal sequence (SS) , the N terminal domain (NTD), the receptor 
binding domain (RBD, which binds the human cellular receptor, ACE -2), subdomain 1 (SD1), and subdomain 2 
(SD2) .The furin cleavage site (S1/ S2) separates S1 from the S2 fragment, which contains the S2 protease 
cleavage site (S2 ′) followed by a fusion peptide (FP), heptad repeats (HR1 and HR2) ,a central helix (CH) 
domain, the connector domain (CD), the transmembrane domain (TM) and a cytoplasmic tail (CT) . 
Source: modified from Wrapp et al, 2020 .
BNT162b2 (V9) encodes a full -length P2 S transmembrane protein that contains two 
consecutive prolines introduced at amino acid positions 986 and 987, between the central 
helix (CH) and heptad repeat 1 (HR1) ( Figure 2.4.2-1 ) (Wrapp et al, 2020; 
Pallesen etal,2017). Two codon optimized forms of the coding sequence for this antigen 
were tested preclinically  and weredesignated “variant 8” and “variant 9” (V8 and V9), with 
the vaccine candidate tested clinically  and being proposed for licensure or authorization, V9, 
expressed from a codon optimized RNA gene with a higher content of cy tosine 
ribonucleotides for increased protein expression. The RNA -expressed P2 S is membrane 
bound and elicits a potent humoral neutralizing antibody  response and Th1 -type CD4+and 
CD8+cellular response to block virus infection and kill virus infected cells, respectivel y.
Efficient in vitro expression of the P2 S protein was demonstrate d following in vitro 
transfection of cells with BNT162b2 RNA drug substance and BNT162b2 drug product.
Electron cryomicroscopy anal ysis of purified recombinant P2 S, expressed from DNA 
encoding the same S amino acid sequence as BNT162b2 RNA (except for the addition of a 
C-terminal tag for protein purification) revealed high similarity  to previously  reported 
structures ( Cai et al, 2020 ). The well -resolved trimeric prefusion structure and the high 
affinity  binding to ACE2 and human neutralizing antibodies dem onstrate that the 
recombinant full -length P2 S protein authentically  presents the ACE -2binding site.
2.4.2.1.3. Immunogenicity of BNT162b2 (V9) in Mice
BNT162b2 was highl y immunogenic in mice with strong antigen -binding IgG and high titer 
neutralizing antibod y respo nses together with a Th1-phenot ype CD4+response as well as an
IFN+, IL-
2+CD8+T-cell response after a single immunization .Total IgG ELISA showed 
that the vaccine induced a strong, dose -dependent IgG response that recognizes S1 and the 
RBD and elicited high neutralizing titers in a pseudoty
pe neutralization assay .
Stimulation of fresh splenocytes, collected 28 days after immunization, with an S protein 
specific overlapping peptide pool demonstrated robust CD4+and CD8+T-cell IFNγ 
responses and a Th1- dominant profile was demonstrated in quantification of cy tokines 
(IL-2and IFNγ) in the corresponding culture supernatants.
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In summary , BNT162b2 induced a strong, neutralizing antibody  response. CD4+and CD8+
T-cell responses were detectable 12 and 28 day s after one immunization and exhibited a 
Th1- dominant T cell response characteristic of RNA -based vaccines .
2.4.2.1.4. Evaluation of BNT162b2 (V9)Immunogenicity and Protection Against 
SARS-CoV-2 Challenge in Rhesus Mac aques
BNT162b2 was assessed for immunogenicity  and for protection against an infectious 
SARS -CoV -2 challenge in rhesus macaques. SARS- CoV -2 infection in humans manifests as 
both asy mptomatic infection and as the disease COVID- 19, with diverse signs, s ymptoms, 
and levels of severit y. Based on published reports, SARS -CoV -2 challenged rhesus 
macaques develop an acute, transient infection in the upper and lower respiratory  tract and 
have evidence of viral replication in the gastrointestinal tract, similar to h umans 
(Zou et al, 2020 ; Kim et al, 2020 ). Vary ing degrees of pulmonary  inflammation, primarily  at 
the peak of infection at approximately  Day  2 to 4 post -challenge, have been reported in the 
literature ( Munster et al, 2020). The human and rhesus ACE -2 receptor have 100% amino 
acid identity  at the critical binding residues, which may  account for the fidelity  of this 
SARS -CoV -2 animal model ( Zhou et al, 2020).
2.4.2.1.4.1. Immunogenicity in Rhesus Macaques
Rhesus macaques immunized I M with 30 µg or 100 µg of BNT162b2 on Day s 0 and 21 had 
readil y detectable S1- binding IgG and SARS -CoV -2 neutralizing titers (NT50) as earl y as 
14days after a single immunization, with substantial increases following the second 
immunization. On Day  28, seven day s after Dose 2 at the 30 µg dose level, the neutralizing 
geometric mean titer (GMT) reached 8 -fold the GMT of a 38 member panel of human 
convalescent sera (HCS); at the 100 µg dose level, the neutralizing GMT was 18- fold the 
HCS GMT. The HCS sera were drawn from SARS- CoV -2 infected i ndividuals 18 to 
83years of age, at least 14 day s after PCR -confirmed diagnosis and at a time when 
individuals were as ymptomatic. The HCS panel provides a currentl y accessible benchmark to 
judge the quality  of the humoral immune response to the vaccine. A decline of b oth,
S1-binding IgG levels and neutralizing titers , was observed out to the latest measured time 
point (Day  56) but remained above the neutralizing GMT and the S1- binding geometric 
mean concentration (GMC) of the HCS.
As seen following mouse i mmunization, strong S -specific Th1- dominant IFNγ+T-cell 
responses were detected in all immunized rhesus macaques . By intracellular cy tokine 
staining anal ysis, there was a dose-dependent increase in S -specific CD4+T cell response s
with a strong Th1 -bias evidenced by  high frequency  of IFNγ+, IL-2+, orTNF -α+cells. 
Notably , CD8+T-cell responses were alsodetectable in BNT162b2- immunized animals .
2.4.2.1.4.2. SARS-CoV-2 Challenge of BNT162b2 (V9) -Immunized Nonhuman Primates
Groups of 2- 4 year old male rhesus macaques that had received two IM immunizations with 
100 µg BNT162b2 V9 (n=6) or saline (Control; n=3) 21 day s apart were challenged 55 day s 
after the second immunization with 1.05 × 106plaque forming units of SARS- CoV -2 (strain 
USA -WA1/2020), split equally  betwee n the intranasal (IN) and intratracheal (I T) routes, as 
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previously  described ( Singh et al, 2020) ( VR-VTR-10671) . SARS -CoV -2 RNA was 
measured b y reverse transcription -quantitative poly merase chain reaction (RT -qPCR) in 
bronchoalveolar lavage fluid, nasal s wabs, and orophary ngeal swabs. The difference in viral 
RNA detection in BAL fluid between BNT162b2 -immunised and control -immunised rhesus 
macaques after challenge is highly  statisticall y significant (b y a nonparametric test, 
p=0.0014). None of the challenged animals showed clinical signs of significant illness, 
indicating that the 2 -4 years old male rhesus challenge model is primaril y an infection model 
for SARS -CoV -2, not a COVID -19 disease model . No radiographic or histological evidence 
of vaccine -elicite d enhanced disease was observed. In summary , BNT162b2 provided 
complete protection from the presence of detectable viral RNA in the lungs compared to the 
saline control with no evidence of vaccine -elicited disease enhancement .
2.4.2.1.5. Immunogenicity Testing AfterWeeklyImmunization of Rats in GLP 
Compliant Repeat Dose Toxicology Studies and a Developmental and Reprodu ctive 
Toxicity Study
The nonclinical safet y data package consists of two GL P-compliant repeat -dose rat toxicity  
studies ,in which both BNT162b2 variants (V8 and V9) were evaluated , anda DART study , 
in which BNT162b2 (V9) was evaluated (Section 2.4.4 ). In allstudies, Study  38166
(evaluating V8) as well as Study  20GR142 andStudy  20256434 (evaluating V9) , the vaccine 
candidates were immunogenic.
In Study  38166, male and female rats received three weekl y IM doses of BNT162b2 (V8). 
Serum samples were collected from main study  animals on Day  17 (two day s after the third 
dose)at the end of the dosing phase and on Day  38 at the end of a 3 -week recovery phase.
The sera were analy zed by  ELISA for IgG that bound S1 and RBD as well as for 
SARS -CoV -2-S pseudovirus neutralizing antibodies. The vaccine candidates elicited IgG that 
recognized S1 and RBD. After immunization, animals developed high titers of 
antigen -specific antibodies as well as pseudovirus neutralization titers.
In Stud y 20GR142, male and female rats received three weekl y IM doses of BNT162b2 
(V9). Serum samples were collected from study  animals prior to vaccine administration, at 
the end of the dosing phase on Day  17 (two day s after the third dose), and at the end of the 
3-week recovery  phase on Recovery  Phase Day  21. Sera were anal yzed for SARS -CoV -2 
neutralizing antibodies. After immunization, BNT162b2 (V9) elicited SARS -CoV -2 
neutralizing antibody  responses in males and females at the end of the dosing and recovery  
phases of the stud y. SARS -CoV -2 neutralizing antibody  responses were not observed in 
animals prior to vaccine administration or in saline -administered control animals.
In Stud y 20256434, female rats were administered 4 total I M doses of BNT162b2 (V9) 21 
and 14 day s prior to mating and on GD9 and GD 20. Serum samples were collected from 
females prior to vaccine administration, just prior to mating (M0), at the end of gestation 
(GD2 1), and at the end of lactation (LD21) and offspring (fetuses on GD21 and pups on 
PND21) . Sera were anal yzed for SARS -CoV -2 neutralizing antibodies. After immunization, 
SARS -CoV -2 neutralizing titers were detected in all maternal females as well as in thei r 
offspring (fetuses and pups) . SARS -CoV -2 neutralizing antibody  titers were not observed in 
animals prior to vaccine administration or in saline -administered control animals.
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2.4.2.2. Secondary Pharmacodynamics
No secondary  pharmacody namics studies were conducted with BNT162b2 .
2.4.2.3.Safety Pharmacology
No safet y pharmacology studies were conducted with BNT162b2 as they  are not considered 
necessary  for the development of vaccines according to the WHO guideline ( WHO, 2005).
2.4.2.4.Pharmacodynamic Drug Interactions
Nonclinical studies evaluating pharmacod ynamic drug interactions with BNT162b2 were not 
conducted as they  are generall y not considered necessary to support development and 
licensure of vaccine products for infectious diseases ( WHO, 2005).
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2.4.3.PHARMACOKINETICS
2.4.3.1.Brief Summary
Assessment of the ADME profile of BNT162b2 (BioNTech code number BNT162, Pfizer 
code number PF -07302048) included evaluating the PK and metabolism of two novel lipid 
excipients (AL C-0315 and AL C-0159) in the LNP and potential biodistribution of 
BNT162b2 using luciferase expression as a surrogate reporter. The luciferase reporter was 
used as it was a readil y available reporter that has been widel y used to de velop an 
understanding of protein /organ expression (Chen et al, 2020; Elia et al, 2020; Fukuchi et al, 
2020; Hassett et al, 2019; Truong et al, 2019; Barry et al, 2012; Jeon et al, 2006 ). An 
intravenous rat PK stud y, using LNPs with the identical lipid com position as BNT162b2, 
demonstrated that ALC -0315 and AL C-0159 distribute from the plasma to the liver. While 
there was no detectable excretion of either lipid in the urine, the percent of dose excreted 
unchanged in feces was ~1% for ALC -0315 and ~50% for A LC-0159. 
The biodistribution of BNT162b2 was evaluated using luciferase expression as a surrogate 
reporter in BALB/c mice. Mice were administered a luciferase expressing modRNA 
formulated like BNT162b2, with the identical lipid composition. Luciferase exp ression was 
measured in vivo following luciferin application. Luciferase expression was identified at the 
injection site at 6 h ours after injection and was not detected after 9 day s.Expression in the 
liver was also present to a lesser extent at 6 hours af ter injection and was not detected by 
48hours after injection. The distribution was also examined in male and female Wistar Han 
rats using LNP swith a comparable lipid composition to BNT162 b2 but with a surrogate 
luciferase RNA and containing trace amounts of radiolabe led [3H]-CHE, a 
non-exchangeable, non -metabolizable lipid marker. The greatest mean concentration of LNP
was found remaining in the injection site in both sexes. Total recovery  (% of injected dose) 
of LNP outside the injection site was greatest in the liver and was much less in the spleen, 
adrenal glands ,and ovaries. 
The in vitro metabolism of AL C-0315 and ALC -0159 was evaluated in blood, liver 
microsomes, S9 fractions, and hepatocy tes from mice, rats, monkey s, and humans. The in 
vivo metabolism was examined in rat plasma, urine, feces, and liver samples from the PK 
study . Metabolism of ALC -0315 and AL C-0159 appears to occur slowl y in vitro and in vivo. 
ALC-0315 and ALC -0159 are metabolized by  hydroly tic metabolism of the ester and amide 
functionalities, respectively , and this hy drolytic metabolism is observed across the species 
evaluated.
In summary , the nonclinical ADME studies indicate that the LNP distributes to the liver. 
Approximately  50% of ALC -0159 is excreted unchanged in feces, while m etabolism play ed 
a role in the elimination of ALC-0315 .
2.4.3.2.Methods of Analysis
No methods of anal ysis have been validated to support GL P TK studies of components of 
BNT162b2 ; however, a qualified L C/MS method was developed to support quantitation of 
the two novel LNP excipients for the non -GLP IV PK study  in rats 
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(Study PF -07302048_06Jul20_072424). Methods for immunogenicity and efficacy studies 
are described in Section 2.6.2.12 .
2.4.3.3.Absorption
2.4.3.3.1. In Vitro Absorption
No absorption studies were conducted for BNT162b2, as the administration route is I M.
2.4.3.3.2. Single-Dose Pharmacokinetics
An intravenous rat PK study  (PF-07302048_06Jul20_072424; Tabulated Summary  2.6.5.3 ) 
was performed using LNP scontaining surrogate luciferase RNA, with the identical lipid 
composition as BNT162b2. This study  was conducted to explore the disposition of 
ALC-0315 and ALC -0159 that had reached the s ystemic circulation following IM 
administration; thus, the IV route was felt to be appropriate . The findings are depicted in
Table 2.4.3-1 andFigure 2.4.3-1.
Table2.4.3-1.  PK of ALC -0315 and ALC -0159 in Wistar Han Rats After IV 
Administration of LNPs Containing Surrogate Luciferase RNA at 1 mg/kg
Analyte Dose of 
Analyte 
(mg/kg)Gender /N t½ 
(h)AUCinf
(µg•h/mL)AUClast
(µg•h/mL)Estimated fraction of 
dose distributed to liver 
(%)a
ALC -0315 15.3 Male/3b139 1030 1020 60
ALC -0159 1.96 Male/3b72.7 99.2 98.6 20
a.Calculated as highest mean amount in the liver (µg)/total mean dose (µg) of ALC -0315 or ALC -0159.
b. 3 animals per timepoint; non-serial sampling.
Figure 2.4.3 -1.Plasma and Liver Concentrations of ALC -0315 and ALC- 0159 in Wistar 
Han Rats After IV Administration of LNP sContaining Surrogate Luciferase RNA at 
1mg/kg
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Pharmacokinetic studies have not been conducted with BNT162b2 and are generall y not 
considered necessary  to support the development and licensure of vaccine products for 
infectious diseases ( WHO, 2005; WHO, 2014).
2.4.3.4.Distribution
In an in vivo study  (R-20-0072 ; Tabulated Summary  2.6.5.5A ), biodistribution was assessed 
using luciferase as a surrogate marker protein, with RNA encoding luciferase formulated like 
BNT162b2, with the identical lipid composition . The LNP -formulated luciferase- encoding 
modRNA was administered to BALB/c mice b y IM injection of 1 µg each in the right and 
left hind leg (for a total of 2 µg). Using in vivo bioluminescence after injection of luciferi n 
substrate, l uciferase protein expression was detected at different timepoints at the site of 
injection and to a lesser extent , and more transiently, in the liver ( Figure 2.4.3-2. Distribution 
to the liver is likely mediated by  LNPs entering the blood str eam. The luciferase expression 
at the injection sites dropped to background levels after 9 day s.The repeat -dose toxicity  
study  in rats showed no evidence of liver injury  (Section 2.4.4.3 ).
The biodistribution of the antigen encoded b y the RNA component of BNT162b2 is expected 
to be dependent on the LNP distribution and the results presented should be representative 
for the vaccine RNA platform , as the LNP -formulated luciferase-encoding modRNA had the 
same lipid composition. 
Figure 2.4.3 -2. Bioluminescence Emission in BALB/c M iceafter IM Injection of an LNP 
Formulation of modRNA Encoding Luciferase
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The distribution of a LNP with a comparable lipid composition to BNT162b2 but with a 
surrogate luciferase RNA (monitoring the 3H-CHE lipid l abel), was investigated in blood, 
plasma and selected tissues in male and female Wistar Han rats over 48 hours after a single 
IMinjection at 50 µg mRNA/animal (Study  185350 ;Tabulated Summary  2.6.5.5B ). The 
greatest mean concentration of LNP was found rem aining in the injection site at each time 
point in both sexes. Outside the injection site, low levels of radioactivity  were detected in 
most tissues, with the greatest levels in plasma observed 1- 4 hours post -dose. Over 48 hours, 
the L NP distributed mainly to liver, adrenal glands, spleen and ovaries, with maximum 
concentrations observed at 8 -48hours post -dose. Total recovery  (% of injected dose) of LNP , 
for combined male and female animals, outside of the injection site was greatest in the liver 
(up to 18%) and was much less in the spleen ( ≤1.0%), adrenal glands ( ≤0.11%) and ovaries 
(≤0.095%). The mean concentrations and tissue distribution pattern were broadly  similar 
between the sexes.
2.4.3.5.Metabolism
Of the four lipids used as excipients in the L NPformulation, two are naturally  occurring 
(cholesterol and DSPC) and will be metabolized and excreted like their endogenous 
counterparts. The in vitro metabolic stability  of the two novel lipids, AL C-0315 (aminolipid) 
and AL C-0159 (PEG- lipid), were evaluat ed in mouse, rat, monkey , and human liver 
microsomes, S9 fractions, and hepatocy tes. AL C-0315 and AL C-0159 were stable (>82% 
remaining) over 120 min in liver microsomes and S9 fractions and over 240 min in 
hepatocy tes in all species and test sy stems ( Studi es 01049-20008, 01049- 20009 , 
01049 -20010 , 01049 -20020 , 01049 -20021 , and 01049 -20022 ;Tabulated Summaries 
2.6.5.10A and 2.6.5.10B ). 
Further stud y of the metabolism of AL C-0315 and AL C-0159 in vitro and in vivo evaluating 
the plasma, urine, feces, and liver from the rat PK study  (Section 2.4.3.3.2 ) determined 
ALC-0315 and ALC -0159 are metabolized slowl y (Study  PF-07302048_05Aug20_043725;
Tabulated Summar ies2.6.5. 9, 2.6.5. 10C,and 2.6.5.10D ). ALC -0315 and AL C-0159 
underwent h ydrol ytic metabolism of the ester and amide functionalities, respectivel y, and 
this hy drolytic metabolism was observed across the species eval uated ( Figure 2.4.3 -3and 
Figure 2.4.3-4 ).
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Figure2.4.3-3. Proposed Biotransformation Pathway of ALC -0315 in Various Species
Metabolism of AL C-0315 occurs via two sequential ester h ydrol ysis reactions, first y ielding 
the monoester metabolite ( m/z528) followed b y the doubly  deesterified metabolite ( m/z290). 
Subsequent metabolism of the doubly  deesterified metabolite resulted in a glucuronide 
metabolite ( m/z466), which was onl y observed in urine from the rat PK study . Additionall y, 
6-hexyldecanoic acid ( m/z255), the acid product of both hy drolysis reactions of AL C-0315, 
was identified.  
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Figure 2.4.3 -4. Proposed Biotransformation Pathway of ALC -0159 in Various Species
The primary  route of metabolism identified for ALC -0159 involves amide bond hy drolysis 
yielding N,N-ditetradecy lamine (m/z410).   
The p rotein encoded b y the RNA in BNT162b2 is expected to be proteol ytically  degraded 
like other endogenous proteins. RNA is degraded by  cellular RNases and subjected to nucleic 
acid metabolism. Nucleotide metabo lism occurs continuously within the cell, with the 
nucleoside being degraded to waste products and excreted or recycled for nucleotide 
synthesis. Therefore, no R NA or protein metabolism or excretion studies will be conducted.
2.4.3.6.Excretion
In the rat PK stud y (Section 2.4.3.3.2 ),there was no detectable excretion of ALC-0315 and 
ALC-0159 in urine after IV administration of LNP scontaining surrogate luciferase RNA at 
1mg/kg. Thepercent excreted unchanged in feces was ~1% for ALC- 0315 and ~ 50% for 
ALC-0159. Metabolites of AL C-0315 were detected in the urine of rats ( Figure 2.4.3 -3). No 
excretion studies have been conducted with BNT162b2 for the reasons described in 
Section 2.4.3.5 .
2.4.3.7.Pharmacokinetic Drug Interactions
No PKdrug interaction studies have been conducted with BNT162b2 .
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2.4.4.TOXICOLOGY
2.4.4.1.Brief Summary
The nonclinical toxicity  assessment of BNT162b2 ( BioNTech code number BNT162, Pfizer 
code number PF -07302048) includes 2 GLP -compliant repeat -dose toxicity  studies and a 
developmental and reproductive toxicity  (DART) study  in Wistar Han rats , outlined below in 
Table 2.4.4 -1. The nonclinical safet y evaluation included 2 variants of BNT162b2: V8 and 
V9. BNT162b2 (V9), the candidate submitted for licensure, differs from BNT162b2 (V8) 
only in the presence of optimized codons to improve antigen expression, but the amino acid 
sequences of the encoded antigens are identical. Two GL P rep eat-dose toxicity  studies for 
BNT162b2 (V8) and BNT162b2 (V9), one stud y for each variant, have been completed. In 
both studies, the nonclinical toxicology findings were similar between BNT162b2 (V9) and 
BNT162b2 (V8). BNT162b2 (V9) was assessed for develo pment and reproductive toxicity  in 
rats.
The IM route of exposure was selected as it is the intended route of clinical administration. 
The selection of rats as the toxicology  test species is consistent with the WHO guidance 
documents on nonclinical evaluat ion of vaccines ( WHO, 2005), which recommend that 
vaccine toxicity  studies be conducted in a species in which an immune response is induced 
by the vaccine. Generation of an immune response to BNT162b2 has been confirmed in rats 
in both repeat -dose toxicity studies and the DART study . The Wistar Han rat is used 
routinely  for regulatory  toxicity  studies, and there is an extensive historical safet y database 
on this strain of rat.
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Table 2.4.4 -1.  Overview of Toxicity Testing Program
StudyaStudy
(Sponsor)
No.Group/
Dose, µg RNATotal 
Volume
(µL)bNo. of 
Animals/
GroupStudy
Status
Repeat-Dose Toxicity
17-Day, 2 or 3 Dose
(1Dose/Week) IM Toxicity 
With a 3 Week Recovery 
Phase in Ratsc,d38166 Controle, 0
BNT162b2 (V8)i, 
100200f
200f15/sex
15/sexCom pleted
17-Day, 3 Dose
(1 Dose/Week)
IM Toxicity With a
3 Week Recovery Phase in 
Ratsg20GR142 Salineh, 0
BNT162b2 (V9)i, 3060
6015/sex
15/sexCom pleted
Developm ental and Reproductive 
Toxicity
Com bined Fertility and 
Developmental Study 
(Including Teratogenicity 
and Postnatal 
Investigations) by the IM 
route in Ratsj20256434
(RN9391
R58)Salineh, 0
BNT162b2 (V9)i, 3060
6044F
44FCom pleted
a. All studies are GLP -compliant and were conducted in an OECD mutual acceptance of data -compliant 
member state.
b. Doses w ere administered as 1 application at 1 site unless otherwise indicated.
c. Study also evaluated the BNT162a1, BNT162b1, and BNT162c1 vaccine candidates.
d. QW x3 (Days 1, 8, 15) for BNT162a1, BNT162b1, and BNT162b2 (V8); QW x2 (Days 1,8) for 
BNT162c1.
e. Phosphate buffered saline, 300 mM sucrose.
f. One application (100 µL) at 2 sites for a total dose volume of 200 µL.
g. Study also evaluated BNT162b3.
h. Sterile saline (0.9% NaCl).
i. BNT162b2 (V8) an d BNT162b2 (V9) both encode the same amino acid sequence of the spike protein 
antigen with tw o prefusion conformation -stabilizing amino acids in the stalk.
j. Study also evaluated BNT162b1 and BNT162b3.
Administration of BNT162b2 by  IM injection to male and female Wistar Han rats once every  
week for a total of 3 weekly  cycles of dosing was tolerated without evidence of s ystemic 
toxicity  in GL P-compliant repeat -dose toxicity  studies. Expected inflammatory  responses to 
the vaccine were evident such as edema and ery thema at the injection sites, transient 
elevation in body  temperature, elevations in WBCs and acute phase reactants, and lower A:G 
ratios. A transient elevation in GGT was noted in animals vaccinated with BNT162b2 (V8) 
in Study  38166 without eviden ce of microscopic changes in the biliary  system or other 
hepatobiliary  biomarkers but was not recapitulated in Study  20GR142. Injection site 
reactions were common in all vaccine -administered animals and were greater after boost 
immunizations. Changes secon dary to inflammation included slight and transient reduction in 
body  weights and transient reduction sin RETI C, PL T, and RBC mass parameters. All 
changes in clinical pathology  parameters and acute phase proteins were reversed at the end of 
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the recovery  phase for BNT162b2 with the exception of higher RDW , higher globulins, and 
lower A:G ratios in animals administered BNT162b2 (V9). The higher RDW reflects prior 
RETI Cincreases. The higher A:G is due to low magnitude increases in globulins, wh ich is an 
expected immune response to vaccine administration ( Sellers et al, 2020 ).
Macroscopic pathology  and organ weight changes were also consistent with immune 
activation and inflammatory  response and included increased size of draining iliac l ymph 
nodes and increased size and weight of spleen. Vaccine-related microscopic findings at the 
end of the dosing phase consisted of edema and inflammation in injection sites and 
surrounding tissue s; increased cellularit y in the draining (iliac)lymph nodes, bone marrow, 
and spleen; and hepatocyte vacuolation in the liver. P eriportal vacuolation of hepatocy tes 
was not associated with any  microscopic evidence of hepatic injury  or alterations in liver 
function tests and is interpreted to reflect hepatocyte uptake of the LNP lipids 
(Sedic etal,2018 ). Microscopic findings at the end of the dosing phase were partiall y or 
completely  recovered in all animals at the end of the recovery  phase for BNT162b2. A robust 
immune response was elicited to the BNT162b2 antigen. 
In the DART study , administration of BNT162b2 to female rats twice before the start of 
mating and twice during gestation at the human clinical dose (30 µg RNA/dosing day ) was 
associated with non adverse effects (bod y weight, food consumption, and localized eff ects in
the injection site) after each dose administration. There were no effects of BNT162b2 
administration on mating performance, fertility , or any  ovarian or uterine parameters in the 
F0female rats nor on embry o-fetal or postnatal survival, growth, or development in the 
F1offspring through the end of lactation. A SARS -CoV -2 neutralizing antibody  response to 
the vaccine was confirmed in F0 female rats prior to mating, at the end of gestation, and at 
the end of lactation and these neutralizing antibodies were also detectable in the F1 offspring 
(fetuses and pups).
2.4.4.2.Single-Dose Toxicity
A separate single -dose toxicity  study  with BNT162b2 has not been conducted.
2.4.4.3.Repeat-Dose Toxicity
2.4.4.3.1. Repeat-Dose Toxicity Study of Three LNP- Formulated RNA Platforms 
Encoding for ViralProteins by Repeated Intramuscular Administration to Wistar Han 
Rats
Thevaccine candidate BNT162b2 (V8), an LNP- formulated modified RNA vaccine 
expressing SARS -CoV -2 P2 S, was assessed in a GL P-compliant repeat -dose toxicity  study  
in Wistar Han rats ( Study 38166 ). This study  also included assessment of 3 other 
LNP -formulated RNA vaccines, encoding either the SARS -CoV -2 P2 S or RBD antigens, 
which were not selected for licensure . For the purpose of this submission, only  the study  
findings from the 100 µg BNT162b2 (V8) vaccine group are summarized; findings from the 
other vaccine candidates were generally  similar.
Administration of BNT162b2 (V8) via IM injections once weekl y for 3 administrations to 
male and female Wistar Han rats was tolerated without evidence of s ystemic toxicity . The 
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vaccine elicited a robust antigen -specific immune response and produced nonadverse 
macroscopic changes at the injection sites, spleen, and t he draining l ymph nodes; increased 
hematopoiesis in the bone marrow and spleen; periportal hepatocy te vacuolation; and clinical 
pathology  changes consistent with an immune response. The findings in this study  were full y 
recovered or showed evidence of ongo ing recovery  at the end of the 3 -week recovery  phase, 
and were consistent with those ty pically  associated with the I M administration of LNP -
encapsulated mRNA vaccines ( Hassett et al, 2019 ).
Body weights were lower 24 hours after each BNT162b2 (V8) vaccine administration 
compared with predose values (down to 0.92x versus baseline) with evidence of weight gain 
(1.22x to 1.37x versus baseline) b y the end of recovery . Bod y weight gain between the 
administrations was comparable to the buffer control group. There were no noteworthy  
effects on bod y weight at the end of the recovery phase. There were no effects on food 
consumption.
BNT162b2 (V8) -administered animals generally  had higher body  temperatures compared 
with buffer control animals at 4 and 24 hours postdos e. Group mean temperatures in rats 
administered the BNT162b2 (V8) vaccine were higher, but within approximately  1°C of the 
group mean bod y temperature of buffer -administered animals. Individual rats administered 
BNT162b2 (V8) did not have bod y temperatures >40.0°C after administration.
Local reactions were observed in male and female animals dosed IM with BNT162b2 (V8). 
The incidence and severity  of the reactions were higher after the second or third injections 
compared with the first injection. The majorit y of animals had very slight edema or rarel y 
slight ery thema after the first dose. After the second or third dose, the severity  of edema and 
erythema increased up to moderate or rarel y, severe grades. These observations resolved 
prior to the next injection or for recovery  animals resolved during the 3- week recovery  phase.
Most BNT162b2 (V8) -related changes in clinical pathology  were consistent with an acute 
phase response and anticipated inflammation. Minor and variable alterations in other clinical 
patholo gy parameters were considered secondary  effects of vaccination.
Expected immune responses to BNT162b2 (V8) were evident in hematology , such as 
elevations in mean neutrophil (up to 7.8x) eosinophil (up to 5.1x controls), basophil (1.47x 
controls) ,and LUC c ounts (up to 7.7x controls) and were highest on Day 17, 48 hours after 
the last injection. WBCs were higher (up to 2.2x controls) in the BNT162b2 (V8) vaccinated 
group on Day 17. PLTcounts were slightly  decreased on Day 17 (down to 0.66x controls). A 
transient reduction in RETI C counts (down to 0.28x controls) was only  observed after the 
administration of the first dose on Day 4. Decreased RETI Cs were similarl y observed in rats 
treated with the licensed LNP -siRNA pharmaceutical Onpattro™ (NDA # 210922) but have 
not been observed in humans treated with this biotherapeutic ( Kozauer et al, 2018), 
suggesting this is a species- specific effect. A slight reduction in red blood cell mass (HGB 
down to 0.87x controls) was observed on Day 17. RETI C and RBC mass parame ter 
decreases were likel y secondary  to the inflammation .
BNT162b2 (V8) -related changes in clinical chemistry  included slightl y higher GGT (a 
biomarker of biliary  and not hepatocellular injury  [Boone et al, 2005 ]) on Day s4 (up to 4.6x 
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controls) and 17 (up to 4.2x controls) without evidence of microscopic changes in the biliary  
system or other hepatobiliary  biomarkers. Additionally , higher GGT was not observed in the 
second repeat -dose toxicity  study  (20GR142), conducted with the clinical candidate 
submitted for licensure. Albumin was slightly  lower on Day s4 (down to 0.87x controls) and 
17 (down to 0.89x controls) and globulin slightl y higher on Day 17 (up to 1.2x controls). 
This resulted in the A:G ratio being slightl y lower on Days 4 (down to 0.84x control s) and 17 
(down to 0.76x controls). The effect on albumin and globulin were related to the vaccine -
mediated inflammatory  response as part of the negative and positive acute phase response, 
respectivel y (Sellers et al, 2020 ).
The acute phase proteins alpha -1-acid gl ycoprotein (up to 21x controls on Day 17) and alpha -
2-macroglobulin (up to 217x controls on Day 17) were elevated in both males and females in 
the BNT162b2 (V8) -administered group on Day s 4 and 17. Fibrinogen was higher in the 
vaccine -administered group (up to 3.1x controls), consistent with an acute phase response. 
Higher concentrations of acute phase proteins are an anticipated response to vaccination.
All changes in clinical pathology  parameters and acute phase proteins were reversed at the 
end of the recovery  phase.
Compared with the buffer control, there were no test -article related differences in the 
concentration of serum cytokines evaluated, in urinaly sis parameters, or in ophthalmoscopic 
or auditory  parameters.
BNT162b2 (V8) -related higher absolute and relative (to body ) spleen weights (up to 1.62x 
controls) were evident and correlated with the macroscopic observation of increased spleen 
size and the increased hematopoiesis. This is likely secondary  to immune responses induced 
by the BNT162b2 (V8) vaccine.
The most common macroscopic observation in the BNT162b2 (V8) group was a thickened 
injection site and/or induration noted for nearl y all animals (16/20) at necropsy . This finding 
correlated with microscopic inflammation at the injection sit e. Macroscopic findings at the 
injection site were resolved at the end of the recovery  phase. Enlarged spleen and iliac ly mph 
nodes were noted in several animals in the BNT162b2 (V8) -administered group , which 
correlated microscopically  to expansion of ly mphoid and/or hematopoietic cells. The effects 
on the ly mphoid organs are consistent with immune responses to the BNT162b2 (V8).
Vaccine -related microscopic findings at the end of dosing were evident in injection sites and 
surrounding tissues, in the drainin g (iliac) ly mph nodes, bone marrow, spleen, and liver.
The inflammation at the injection site was characterized by  infiltrates of macrophages, 
granulocy tes, and l ymphocy tes into the muscle, and variabl y into the dermis and subcutis. 
Injection site inflammation was associated with moderate edema, mild m yofiber 
degeneration, occasional muscle necrosis, and mild fibrosis. I njection site findings were 
consistent with an immune/inflammatory  response to an IMvaccine administration.
In the draining (iliac) lymph node, increased cellularity  of the follicular germinal centers and 
increased plasma cells (plasmacy tosis) were variably  present for all BNT162b2 (V8) -dosed 
animals. I n addition, minimal to mild increases in the cellularit y of bone marrow and 
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hematopoiesis in the spleen likely  related to increased granulopoiesis and correlated with 
increased circulating neutrophils (which correlated with increased spleen size and weight) 
were present in BNT162b2 (V8) -dosed animals.
Vacuolation of hepatocy tes (minimal to mild ) in the portal regions of the liver were present 
for all BNT162b2 (V8) -dosed animals. The liver findings were not associated with changes 
in markers of hepatocy te injury  (eg, AST or ALT). While GGT was elevated in vaccine -
administered animals, it was not considered to be associated with the vacuolation of 
hepatocy tes (Ennulat et al, 2010 ). The microscopic observation of liver vacuolation is 
believed to be associated with hepatocy te uptake of the LNP lipids ( Section 2.4.3.4 ; 
Sedic etal, 2018 ).
Microscopic findings at the end of the dosing phase were partiall y or completely resolved in 
all animals at the end of the recovery phase. Inflammation at the injection site and 
surrounding tissues was less severe (minimal to mild) in animals administered 
BNT162b2 (V8) at the end of the 3- week recovery  phase, indicating partial recovery . In the 
iliac ly mph node, plasmacy tosis was less severe, and macrophage infiltrates were present at 
the end of the 3- week recovery  phase and reflect resolution of the inflammation note d at the 
end of the dosing phase.
All other observations in the bone marrow, spleen, and liver were full y resolved at the end of 
the 3- week recovery  phase.
The immune response to the vaccine antigen was evaluated b y S1-binding IgG and RBD -
binding IgG ELISAs, and a SARS -CoV -2 S pseudoty pe neutralization (pVNT) assay  on
Days17 and 38 ( Section 2.4.2.1.4 ). The data demonstrate that BNT162b2 (V8) elicited a 
SARS -CoV -2 S- specific antibody  response with high neutralizing activit y.
In conclusion, administrati on of BNT162b2 (V8) by  IM injection to male and female Wistar 
Han rats once every  week for 3 doses, was tolerated at 100 µg RNA /dosing day without 
evidence of s ystemic toxicity .
2.4.4.3.2. 17 -Day Intramuscular Toxicity Study of BNT162b2 (V9) in Wistar Han Rats 
with a3-week Recovery
Thevaccine candidate BNT162b2 (V9), an LNP- formulated modified RNA vaccine 
expressing SARS -CoV -2 P2 S, was assessed in a GL P-compliant repeat -dose toxicity  study  
in Wistar Han rats ( Study 20GR142). This study  also included assessment of a nother LNP -
formulated RNA vaccine candidate (BNT162b3) that will not be included in the licensure 
application. For the purpose of this submission, the study findings from the BNT162b2 (V9) 
vaccine are summarized; findings from the BNT162b3 vaccine candidat e also tested in this 
study  were generally  similar. BNT162b2 (V9) was administered at 30 µg once weekl y for 
3doses (Day s 1, 8, and 15) followed b y a 3 -week recovery  phase.
Administration of BNT162b2 (V9) via IM injections once weekl y for 3 administrations to 
male and female Wistar Han rats was tolerated without evidence of s ystemic toxicity . The 
vaccine elicited a robust antigen -specific immune response and produced nonadverse 
macroscopic changes at the injection sites, spleen, and the draining lymph nodes; increased 
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hematopoiesis in the bone marrow and spleen; liver vacuolation; and clinical pathology  
changes consistent with an immune response. The findings in this study  were either full y 
recovered or showed evidence of ongoing recovery at the end of the 3 -week recovery  phase, 
and were consistent with those ty pically  associated with the I M administration of LNP -
encapsulated mRNA vaccines ( Hassett et al, 2019 ).
All animals administered BNT162b2 (V9) survived to scheduled necrops y. There were no 
test article -related clinical signs or body  weight changes noted. Test article- related reduced 
mean food consumption was noted on Day s4 and 11 (down to 0.83x controls). Test article -
related higher mean bod y temperature (maximum increase post each dose) compared with 
control animals was noted on Day 1 (up to 0.54°C), Day 8 (up to 0.98°C), and Day 15 (up to 
1.03°C) postdose.
BNT162b2 (V9) -related injection site edema and ery thema were noted on Day s1 (up to 
slight edema and very  slight ery thema), 8 (up to moderate edema and very  slight ery thema), 
and 15 (up to moderate edema and very  slight ery thema). The incidence and severit y of the 
reactions were higher after the second or third injections compared with the first injection. 
Test article -related ery thema and edema full yresolved prior to dose administration on Day s 8 
and 15. Injection site ery thema and edema were fully  resolved at the end of the recovery  
phase .
All clinical pathology  changes (t ype and magnitude) were generally consistent with expected 
immune responses to the vaccine or secondary  to inflammation.
There were higher WBCs (up to 2.95x controls), primarily  involving neutrophils (up to 6. 60x 
controls), monocy tes (up to 3.30x controls), and LUC (up to 13.2x controls) and slightl y 
higher eosinophils and basophils on Day s4 and 17. WBCs were higher on Day 17 as 
compared with Day 4. There were transientl y lower RETICs on Day 4 (down to 0.27x 
controls) in both sexes and higher RETI Cs on Day 17 (up to 1.31x controls) in females onl y. 
Lower RBC mass parameters (down to 0.90x controls) were present on Day s4 and 17. All 
test article -related hematology  and coagulation changes noted in the dosing phase were full y 
reversed after a 3 -week recovery  phase, with the exception of higher red cell distribution 
width (up to 1.21x c ontrols) in animals administered BNT162b2 (V9). The higher RDW 
reflects prior reticulocy te increases.
There were lower A:G ratios (down to 0.82x) on Day s 4 and 17. Higher fibrinogen levels 
were observed on Day 17 (up to 2.49x ) when compared with control animals, consistent with 
an acute phase response. The acute phase proteins alpha-1 -acid glycoprotein (up to 39x on 
Day 17) and alpha -2-macroglobulin (up to 7 1xon Day 17) were elevated in both males and 
females in the BNT162b2 (V9) -administered group on Day s4 and 17 with higher 
concentrations generally  observed in males. All other changes in clinical pathology  
parameters were considered incidental. All test art icle-related clinical chemistry  changes 
noted in the dosing phase were fully  reversed after a 3 -week recovery  phase, except higher 
globulins (up to 1.08x controls) in animals administered BNT162b2 (V9) and lower A:G 
ratio (down to 0.91x controls) in female s administered BNT162b2 (V9), reflecting vaccine -
related immune response.
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Test article -related higher group mean absolute and relative spleen weights (compared to 
body  weight) were noted in males that had received BNT162b2 (V9) (up to 1.42x) and 
females ( up to 1.59x) relative to control group means. There were no other test article -related 
changes in organ weights. At the end of the recovery  phase, spleen weights were within 
normal limits.
Test article -related macroscopic findings included the observation of enlarged draining 
lymph nodes (2/20 animals) and pale/dark (5/20 animals) or firm (6/20 animals) injection 
sites in animals administered BNT162b2 (V9). These changes full y recovered, except for 
partial recovery  of enlarged draining nodes, suggesting rec overy  in progress. 
Test article -related microscopic pathology  findings were observed at the injection site and in 
thedraining (iliac) and inguinal lymph nodes, spleen, bone marrow, and liver for both 
vaccine candidates. All microscopic findings were nona dverse, as there was no evidence of 
systemic toxicity  or clinical signs of illness or lameness. 
At the end of the dosing phase, test article- related mixed cell inflammation (mild to 
moderate) and edema (mild to moderate) at the injection site were consist ent with findings 
typicall y associated with the I M administration of LNP -encapsulated mRNA vaccines 
(Hassett et al, 2019 ). These findings correlated with macroscopic observations of abnormal 
color (dark/pale) and consistency  (firm). At the end of the 3 -week recovery phase, there was 
full recovery  for injection site edema and partial recovery  for injection site inflammation, 
suggesting recovery  in progress.
At the end of the dosing phase, test article- related findings in the draining (iliac) and inguinal
lymph nodes (up to moderatel y increased cellularity of plasma cells and germinal centers), 
spleen (minimally  increased cellularit y of hematopoietic cells and germinal centers), and the 
bone marrow (minimal increased cellularity  of hematopoietic cells) were pr esent. These 
changes are secondary  to immune activation and/or inflammation at the injection site. The 
presence of plasma cells (interpreted as plasmablasts) in the draining (iliac) and inguinal 
lymph nodes is consistent with a robust immunological respons e to the vaccines. These 
observations correlated with macroscopic observations of abnormal size (enlarged) in the 
lymph nodes and spleen and increased spleen weights. At the end of the 3 -week recovery  
phase, full recovery  of increased cellularity  of hemato poietic cells in the spleen and bone 
marrow, with partial recovery  (recovery  in progress) of increased cellularit y of plasma cells 
and germinal centers in the draining and inguinal ly mph nodes, and increased cellularit y of 
the germinal centers in the spleen. 
At the end of the dosing phase, the test article- related microscopic finding of minimal peri -
portal hepatocy te vacuolation was not associated with hepatocellular damage or alterations in 
liver function tests. The liver vacuolation is believed to be ass ociated with hepatocy te uptake 
of the LNP lipids ( Section 2.4.3.4 ; Sedic et al, 2018 ). At the end of 3 -week recovery  phase, 
this finding was completely  recovered. 
Administration of 3 once weekl y doses of BNT162b2 (V9) elicited SARS -CoV -2 
neutralizing antibody  responses in males and females at the end of the dosing (Day  17) and 
recovery  phases (Day  21) of the study . SARS -CoV -2 neutralizing antibody  responses were 
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not observed in animals prior to vaccine administration or in saline -administered control 
anima ls.
In conclusion, administration of BNT162b2 (V9) at 30 µg RNA/dosing day  via IM injections 
weekl y for 3 administrations to male and female Wistar Han rats was tolerated without 
evidence of s ystemic toxicity . Dosing of BNT162b2 (V9) produced changes consi stent with 
an inflammatory  response and immune activation. The findings in this study  are consistent 
with those ty picall y associated with the IM administration of L NP-encapsulated mRNA 
vaccines.
2.4.4.4.Genotoxicity
No genotoxicity  studies are planned for BNT162b2 as the components of the vaccine 
construct are lipids and RNA and are not expected to have genotoxic potential ( WHO, 2005).
2.4.4.5.Carcinogenicity
Carcinogenicit y studies with BNT162b2 have not been conducted as the components of the 
vaccine construct are lipids and RNA and are not expected to have carcinogenic or 
tumorigenic potential. Carcinogenicit y testing is generally not considered necessary to 
support the development and licensure of vaccine products for infectious diseases 
(WHO, 2005 ).
2.4.4.6. Reproductive and Developmental Toxicity
Reproductive and developmental toxicity  assessments were made with BNT162b2 (V9) 
(Study 20256434 ). BNT162b2 was administered by  IM injection at the human clinical dose 
(30µgRNA/dosing day ) to 44 female Wistar Han rats (F0) 21and14days prior to mating 
with untreated males and on GD 9and 20, for a total of 4 dosing day s. A separate control 
group of 44 F0females received saline by  the same route and regimen.
Following completion of a mating phase with untreated males, 22 rats/group underwent 
caesarean- section on GD 21 and were submitted to routine embry o-fetal development 
evaluations. The remaining 22 rats/group were allowed to litterand development of the 
offspring was observed until PND 21.
There were no BNT162b2 -related deaths during the study .IMadministration of BNT162b2
before and during gestation to female Wistar ratsresulted in nonadverse clinical signs and 
macroscopic findings localized to the injection site as well as transient, nonadverse bod y 
weight and food consumpti on effects after each dose administration. These maternal findings 
are all consistent with administration of a vaccine and an inflammatory /immune response.
There were no BNT162b2 -related effects on an y mating or fertility  parameters. There were 
noBNT162b2 -related effects on an y ovarian, uterine, or litter parameters, including embry o-
fetal survival, growth, or external, visceral, or skeletal malformations, anomalies, or 
variations. There were no effects of BNT162b2 administration on postnatal offspring (F1 ) 
development, including postnatal growth, physical development (pinna unfolding and ey e 
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opening), reflex ontogeny (pre-weaning auditory  and visual function tests), macroscopic 
observations, andsurvival.
AllF0females administered BNT162b2 developed SARS -CoV -2neutralizing antibod iesand 
these antibodies were also detectable in all fetuses and pups from the caesarean and littering 
groups, respectively . The animals in the saline control group did not exhibit an immune 
response to BNT162b2.
In conclusion, administration of BNT162b2 to female rats twice before the start of mating 
and twice during gestation at the human clinical dose was associated with nonadverse effects
(body  weight, food consumption ,and effects localized to the injection site) after ea ch dose 
administration. However, there were no effects of BNT162b2 administration on mating 
performance, fertility , or any  ovarian or uterine parameters in the F0 female rats nor on 
embry o-fetal or postnatal survival, growth, or development in the F1 offsp ring. An immune 
response was confirmed inF0female rats following administration of each vaccine candidate 
and these responses were also detectable in the F1offspring (fetuses and pups).
Macroscopic and microscopic evaluation of male and female reproductive tissues from the 
repeat -dose toxicity  studies with BNT162b2 showed no evidence of toxicity .
2.4.4.7.Local Tolerance
Local tolerance of IM administration of BNT162b2 was evaluated by  injection site 
observations and macroscopic and microscopic examinati on of injection sites in the repeat -
dose toxicity  studies and is described in Section 2.4.4.3.
2.4.4.8.Other Toxicity Studies
2.4.4.8.1. Phototoxicity
Phototoxicity  studies with BNT162b2 have not been conducted.
2.4.4.8.2. Antigenicity
Immunogenicity  was evaluated as part of the primary pharmacod ynamic studies 
(Section 2.4.2.1 ). Serology  data from the repeat -dose toxicity  studies shows a robust antigen -
specific immune response to BNT162b2.
2.4.4.8.3. Immunotoxicity
Stand -alone immunotoxicity  studies with BNT162b2 ha ve not been conducted. However, 
immunotoxicological endpoints were collected as part of the repeat -dose toxicity  studies; 
there were no adverse effects observed and no significant effects on measured cy tokines.
2.4.4.8.4. Mechanistic Studies
Mechanistic studies with BNT162b2 have not been conducted.
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2.4.4.8.5. Dependence
Dependence studies with BNT162b2 have not been conducted.
2.4.4.8.6. Studies on Metabolites
Stand -alone studies with administration of metabolites of BNT162b2 have not been 
conducted.
2.4.4.8.7. Studies on Impurities
Stand -alone studies with administration of impurities of BNT162b2 have not been conducted.
2.4.4.8.8. Other Studies
No other studies with BNT162b2 evaluated in this submission have been conducted.
2.4.4.9.Target Organ Toxicity
Based on data from the GL P repeat -dose toxicity  studies ( Section 2.4.4.3 ), administration of 
BNT162b2 was well tolerated without an y evidence of s ystemic toxicity . BNT162b2
administration was associated with local reactogenicity  at the injection site and expected 
inflammatory  responses, including increases in l ymphoid cells in draining ly mph nodes and 
spleen. Microscopic findings within injection sites, which were partiall y reversed b y the end 
of recovery , support this conclusion. The liver finding was reversible, n ot associated with 
changes in markers of hepatocy te injury  and not considered adverse. The elevated levels of 
GGT in Study 38166 were not recapitulated in Study 20GR142 and were not associated with 
hepatobiliary  changes microscopically . Elevated GGT was no t attributed to the hepatocy te 
vacuolation ( Ennulat et al, 2010 ).
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2.4.5. INTEGRATED OVERVIEW AND CONCLUSIONS
The nonclinical program demonstrates that BNT162b2 is immunogenic in mice, rats, and 
nonhuman primates, and the toxicity studies support the licensure of this vaccine. Preclinical 
assessments in mice and nonhuman primates demonstrate that BNT162b2 elicits a rapid 
antibody  response with measurable SARS -CoV -2 neutralizing titers after a single dose and 
substantial increases in titers after a sec ond dose that exceed titers in sera from 
SARS -CoV -2/COVI D-19-recovered patients. A Th1- dominant T cell response was evident in 
both mice and nonhuman primates. In a SARS -CoV -2 rhesus challenge model, 
BNT162b2 provided complete protection in the lungs, as d etermined b y lack of detectable 
viral RNA, and there was no evidence of vaccine -elicited disease enhancement.
An IVrat PK study , using an LNP with the identical lipid composition as BNT162b2 , 
demonstrated that the novel lipid excipients in the L NP formula tion, AL C-0315 and 
ALC-0159 ,distribute from the plasma to the liver. While there was no detectable excretion 
of either lipid in the urine, the percent of dose excreted unchanged in feces was ~1% for 
ALC-0315 and ~ 50% for AL C-0159. Further studies indicate d metabolism played a role in 
the elimination of AL C-0315 . Biodistribution was assessed using luciferase expression as a 
surrogate reporter formulated like BNT162b2, with the identical lipid composition . After IM 
injection of the LNP -formulated RNA encodin g luciferase in BALB/c mice, luciferase 
protein expression was demonstrated at the site of injection 6 h ours post dose and was not 
detected after 9 days. Luciferase was detected to a lesser extent in the liver ; expression was 
present at 6 hours after injection and was not detected by 48 hours after injection. After IM 
administration of a radiolabeled LNP -mRNA formulation containing ALC -0315 and 
ALC-0159 to rats, the percent of administered dose was also greatest at the injection site. 
Outside of the injection site, total recovery of radioactivity  was greatest in the liver and much 
lower in the spleen, with very  little recovery  in the adrenal glands and ovaries. The 
metabolism of AL C-0315 and AL C-0159 was evaluated in blood, liver microsomes, S9 
fractions, and hepatocy tes from mice, rats, monkeys, and humans. The in vivo metabolism 
was examined in rat plasma, urine, feces, and liver samples from the PK study . Metabolism 
of AL C-0315 and ALC -0159 appears to occur slowly  invitro and in vivo. AL C-0315 and 
ALC-0159 are metabolized by  hydroly tic metabolism of the ester and amide functionalities, 
respectivel y, and this hy droly tic metabolism is observed across the species evaluated.
Administration of BNT162b2 by  IM injection to male and female Wistar Han r ats once every  
week for a total of 3 weekly  cycles of dosing was tolerated without evidence of s ystemic 
toxicity  in GL P-compliant repeat -dose toxicity  studies. Expected immune responses to the 
vaccine were evident such as edema and ery thema at the injectio n sites, transient elevation in 
body  temperature, elevations in WBCs and acute phase reactants, and decreased A:G ratios . 
Injection site reactions were common in all vaccine -administered animals and were greater 
after boost immunizations. Changes secondary to inflammation included slight and transient 
reductions in body  weights and transient reduction s in RETI C, PL T, and RBC mass 
parameters. All changes in hematology  parameters and acute phase proteins were similar to 
control at the end of the recovery  phas e for BNT162b2 with the exception of higher RDW 
and lower A:G ratios in animals administered BNT162b2 (V9). Macroscopic pathology  and 
organ weight changes were also consistent with immune activation and inflammatory  
response and included increased size of draining iliac ly mph nodes and increased size and 
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weight of spleen. Vaccine- related microscopic findings at the end of dosing for BNT162b2
were evident in injection sites and surrounding tissues, in the draining iliac ly mph nodes, 
bone marrow, spleen, and liver. Microscopic findings at the end of the dosing phase were 
partially  (recovery  in progress) or completel y recovered in all animals at the end of the 
recovery  phase for BNT162b2. A robust immune response was elicited to the BNT162b2
vaccine antigen.
Administration of BNT162b2 to female rats twice before the start of mating and twice during 
gestation at the human clinical dose (30µg RNA/dosing day ) was associated with 
nonadverse effects (bod y weight, food consumption and effects localized to the injection site) 
after eac h dose administration. There were no effects of BNT162b2 administration on mating 
performance, fertility , or any  ovarian or uterine parameters in the F0 female ra ts nor on 
embry o-fetal or postnatal survival, growth, or development in the F1 offspring. An immune 
response was confirmed in F0 female rats following administration of BNT162b2 and this 
response was also detectable in the F1 offspring (fetuses and pups).
In summary , the nonclinical package summarized above supports the BLA of BNT162b2
administered twice b y IM injection at a dose of 30µg RNA .
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Hassett KJ, Benenato KE, Jacquinet E et al. Optimization of lipi d nanoparticles for 
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Jeon YH, Choi Y, Kang JH, et al. I n vivo monitoring of DNA vaccine gene expression using 
firefl y luciferase as a naked DNA. Vaccine 2006;24(16) :3057 -62.
Jiang S, Hy llyer C, Du L. Neutralizing antibodies against SARS -CoV -2 and other human 
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