docs/Toxicology Review - COMIRNATY.txt

BLA 125742
Toxicology Review of COVID-19 Vaccine (BNT162, PF-07302048)
(Final Report)
From:
Nabil, Al Humadi
Through:
Martin Green
To:
Ramachandra Naik, Michael Smith
File:
BLA 125742, original submission
Product:
COVID-19 Vaccine (BNT162, PF-07302048)
Reviewer: Nabil Al-Humadi
BLA sections reviewed:
4.2.3.2 Repeated dose toxicity studies
4.2.3.5. Reproductive and Developmental Toxicity
Type and date of submission: Original, August 315, 2018
Sponsor: BioNTech RNA Pharmaceuticals GmbH, An der Goldgrube 12 Mainz Germany 55131
Proposed indication: Prophylactic immunization against COVID-19 in adults ≥18 years of age
Division name: OVRR/DVRPA
Table of contents
Proposed indication:
Précis:
Introduction:
Proposed clinical study:
Study number 1
Study number 2:
Study number 3 (Reproductive Toxicology Study):
Historical data:
References:....
10
...... 64
.... 93
.... 117
......... 123
Table of text tables:
Table 1: Protocol of stability study I for CTM drug substance batches at different storage conditions.
Table 2: Experimental design.
Table 3: Blood sampling schedule for laboratory examinations
Table 4: Acute phase proteins.
Table 5: Cytokine analysis..
Table 6: Weighed organs.
Table 7: Serum chemistry results
.... 11
.... 12
.... 13
... 14
14
14
.. 16

BLA 125742
Table 8: Differences in albumin and globulin levels and the albumin/ globulin ratio compared to the control group ....
Table 9: Hematological results .......
.... 20
........ 23
Table 10: Test article-related changes in hematological and coagulation parameters for the treatment with BNT162a1...........
Table 11: Test article-related changes in hematological and coagulation parameters..
..... 25
....... 26
Table 12: Test article-related changes in hematological and coagulation parameters for the treatment with BNT162c1......
.... 27
Table 13: test article-related changes in hematological and coagulation parameters
..... 28
Table 14: Acute phase protein levels, day 4 relatives to start date...
.... 29
Table 15: Acute phase protein levels, day 10 relatives to start date ....
.... 29
Table 16: Acute phase protein levels, day 17 relatives to start date.
. 30
Table 17: Cvtokine levels in males at stud day 1............
.31
Table 18: Cytokine levels in males at study day 8................
....32
Table 19: Cytokine levels in males at study day 15......
....33
Table 20: Cytokine levels in males at study day 17 relatives to start date (48h pa)
.... 34
Table 21: Cvtokine levels in females at study day 1 .....................
.... 35
Table 22: Cytokine levels in females at study day 8 ........
.... 36
Table 23: Cvtokine levels in females at study day 15 ......................
.... 36
Table 24: Cytokine levels in females at study day 17 relatives to start date (48h pa)
....... 37
Table 25: Urinalysis results in males at day 10 relatives to start date.....
...... 37
Table 26: Urinalysis results in males at day 17 relatives to start date ....
....... 38
Table 27: Urinalysis results in females at day 10 relatives to start date....
....... 38
Table 28: Urinalysis results in females at dav 17 relatives to start date....
......... 39
Table 29: Male's organ weights results. Absolute weights are expressed as mean (grams). Entries in table are expressed both as organ weight from animals taken at the end of the terminal phase and recovery phase of the study (main phase organ weight/recovery phase organ weight)......... 43
Table 30: Female's organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed both as organ weight from animals taken at the end of the terminal phase and recovery phase of the study (main phase organ weight/recovery phase organ weight)..
.... 45
Table 31: Male's gross pathology results...
.... 46
Table 32: Female's gross pathology results..
.....46
Table 33: Incidences of test article-related microscopic findings for the animals treated with BNT162a1..
Table 34: Incidences of test article-related microscopic findings for the animals treated.
....48
.... 49
Table 35: Incidences of test article-related microscopic findings for the animals treated with BNT162c1 and BNT16262
Table 36: Microscopic findings at terminal sacrifice
Table 37: Test article related effects.
.... 50
..51
....59
Table 38: Protocol of stability study I for CTM drug substance batches at different storage conditions.....
Table 39: parameters evaluated
Table 40: Clinical laboratory measurements
Table 41: Antibody (Serology) response to vaccine components
Table 42: Tissue collection, organ weights and tissues processed for slide preparation - Dosing phase
.... 65
.... 67
.... 68
.... 68
..... 69
2

BLA 125742
Table 43: Tissue collection, organ weights and tissues processed for slide preparation - Recovery phase .....
.... 71
Table 44: Serum chemistry results for males and females .......
............ 71
Table 45: Test article-related clinical chemistry parameter effects (mean control values and ratio relative to control mean) ......
..... 72
Table 46: Test article-related clinical chemistry parameter effects (mean control values and ratio relative to control mean) .......
....... 72
Table 47: Hematology results for males and females...
•.........….. 7 3
Table 48: Test article-related hematology and coagulation parameter effects at main sacrifice (mean control values and ratio relative to control mean) .........
...... 75
Table 49: Test article-related hematology and coagulation parameter effects at recovery phase (mean control values and ratio relative to control mean) ............
....... 75
Table 50: Male's organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed as organ weight from animals taken at the end of the terminal phase. .................. 76
Table 51: Female's organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed as organ weight from animals taken at the end of the terminal phase........... 77
Table 52: Gross findings at dosing phase...................................
....... 78
Table 53: Macroscopic findings at recovery phase...............
....... 78
Table 54: Microscopic findings at terminal sacrifice ........................
...... 82
Table 55: Edema and erythema findings in males at study days 1, 8, and 15 ....
Table 56: Edema and erythema findings in females at study days 1, 8, and 15 ....
Table 57: Edema and erythema findings in males and females at recovery phase..
Table 58: Urinalysis for male groups...........
5388
Table 59: Urinalysis for male groups..........
Table 60: Geometric mean titers (GMTs) for each dose group by sampling day and sex
Table 61: Test item identification.....
Table 62: Control item identification.........
Table 63: Experimental design of the FO generation...
Table 64: General in-life assessments - untreated males and F0 females....
Table 65: Geometric mean titer by time-point and by group of females or offspring (fetuses and pups)......
Table 66: Mean estrous cycle data - Before dosing...
Table 67: Mean estrous cycle data - Pre-mating period
Table 68: Summary of cohabitation data and maternal performance in littering and Caesarean subsets .......
Table 69: Mean gravid uterus weight and maternal body weight change
Table 70: Mean Caesarean section data.
Table 71: Summary of Foetal External, Visceral and Skeletal Observations Table 72: Delivery and litter data
Table 73: Mean pup body weight (grams)..
Table 74: Summary of reflex and physical development
Table 75: Summary of maternal macroscopic observations.
Table 76: Historical data; Caesarean data collected on day 21 of gestation - page 1/2
Table 77: Historical data; Caesarean data collected on day 21 of gestation - page 2/2
Table 78: Historical data; Malformations (external, internal and skeletal)..
...87
..... 93
..... 93
...... 95
......... 96
...... 99
... 100
....... 100
.... 102
... 103
... 104
. 108
. 110
114
114
115
117
. 118
... 119
3

BLA 125742
Table 79: Historical data; Foetal examination- Fresh visceral examination of body on day 20 or 21 of gestaion....
Table 80: Historical data; Foetal examination - Skeletal examination of body on day 21 of gestation - Page 1/2
Table 81: Historical data; Foetal examination - Skeletal examination of body on day 21 of gestation - Page 2/2.....
Table 82: Historical data; Foetal examination - Skeletal examination of head on day 21 of gestation ....
.... 120
..... 121
... 122
... 123
Table of figures:
Figure 1: BioNTech non-clinical platform experience ..................
.... 6
Figure 2: Summary of vaccine dose regimens in the clinical study ....
..... 8
Figure 3: Part A; Dose cohort scheme for uRNA (BNT162a1) and saRNA (BNT162c1)........... 8
Figure 4: Part A; Dose cohort scheme for modified RNA groups (BNT162b1 and BNT162b2) ..9
Figure 5: Gamma-glutamyltransferase plasma activity in male rats mean values per group and
standard deviation. TD = Treatment day. ........
.......... 17
Figure 6: Gamma-glutamyltransferase plasma activity in female rats mean values per group and
standard deviation. TD = Treatment day. ........
......... 17
Figure 7: Test article-related changes in plasma activity of gamma-glutamyltransferase compared to the control group in % ......
....... 18
Figure 8: Reticulocyte's levels....
....... 24
Figure 9: Local reactions.....
....... 41
Figure 10: Body weight gain of male rats.....
....... 42
Figure 11: Body weight gain of female rats.....
....... 42
Figure 12: Body temperature of male rats treated once weekly, mean values per group............. 57
Figure 13: Body temperature of female rats treated once weekly, mean values per group.......... 57
Figure 14: Antibody titer resulting in 50% pseudovirus neutralization activity (p VN50).
Individual VNT titers resulting in 50% pseudovirus neutralization (pVN50) are shown by dots; group mean values are indicated by horizontal bars (‡SEM, standard error of the mean)........... 58
Figure 15: antibody titer resulting in (b) (4)
pseudovirus neutralization activity (pVN°
Individual VNT titers resulting in (b) (4) pseudovirus neutralization (pVNO
**) are shown by dots;
group mean values are indicated by horizontal bars (‡SEM, standard error of the mean)...
Figure 16: Mean pup bod weights (g)-Males.....
Figure 17: Mean pup body weights (g)-Females
....58
....... 111
111
Précis:
Study number 1:
In this repeat (groups 1 to 5 and 7 animals were dosed by IM on study days 1, 8, and 15 and group 6 animals were dosed on study days 1 and 8) dose toxicology study, rats were assigned to 7 different groups and treated with control or test article (see experimental design). Animals, 18 per sex per group, were treated with a final dose concentration of 0, 10, 30, or 100 [ug/animal].
Animals were euthanized on study davs 10 and 17. Except for group 6 (014 ug/animal [LNP saRNA RBD] test item 5), immune responses were reported in all other treated groups.
Study number 2:
4

BLA 125742
In this repeat (study days 1, 8, and 15) dose toxicology study, rats were assigned to 3 different groups and treated with control or test article (see experimental design). Animals, 15 per sex per group, were treated with a final dose concentration of 30 [ug/animal]. Animals were euthanized on study days 17 and 22. Immune responses were reported in all treated groups.
Study number 3 (Developmental toxicology study):
Animals were randomized and assigned to 4 different groups. Each group consisted of 22 females. Animals were administered 4 doses of saline or test article (30 [ug/animall) on study day 1 (21 days before mating, M-21) and day 8 (14 days before mating, M-14) and on gestation days 9 and 20. Animals were euthanized according to the following schedule:
FO Females: Caesarean subset: On GD21.
Littering subset: After weaning of the F1 pups (females that fail to produce a viable litter by GD26 will be euthanized and necropsied).
Unmated Females: After completion of the mating period.
Pups: On PND4 (unselected pups) or on PND21.
Introduction:
Coronavirus infection 2019 (COVID-19) are increasing every day and spreading globally, affecting more and more countries.
The World Health Organization (WHO) characterized the COVID-19 outbreak as pandemic on March 11th, 2020. At the time of writing this report, more than 15 million people around the world were affected and more than 600 thousand people were died. Currently, no approved vaccines or antiviral drugs to prevent or treat SARS-CoV-2 infections or its associated disease
COVID-2019 (1).
Significant advantage over more conventional vaccine approaches when using an RNA-based vaccine encoding a viral antigen that is translated to protein by the vaccinated organism to induce a protective immune response. RNA vaccines do not carry the risks associated with infection, unlike live attenuated vaccines. This kind of vaccines may be given to people who cannot be administered live virus (such as pregnant women and immunocompromised persons).
The manufacturing of the RNA-based vaccines is via a cell-free in vitro transcription process.
This method allows an easy and rapid production, and the prospect of producing high numbers of vaccination doses within a shorter time period than achieved with conventional vaccine approaches. In outbreak scenarios, this capability is pivotal to enable the most effective response.
The core innovation of the RNA vaccine is based on in vivo delivery of a pharmacologically optimized, antigen-encoding RNA to induce robust neutralizing antibodies and a concomitant T cell response to achieve protective immunization with minimal vaccine doses (2-4).
There are three different RNA platforms under development at BioNTech. These platforms are nonmodified uridine containing mRNA (uRNA, BNT162a), nucleoside modified mRNA (modRNA, BNT162b), and self-amplifying mRNA (saRNA, BNT162c). In more than a dozen non-clinical GLP safety studies, all three RNA platforms have been tested. As for uRNA and modRNA, there is pre-existing clinical safety data. These data have been obtained primarily with
5

BLA 125742
RNAs formulated with (b) (4) which are related, but not identical, to those to be used in this trial.
Generated by BioNTech, the non-clinical toxicity data suggest a favorable safety profile for uRNA and modRNA, as well as saRNA formulated with different nanoparticles for various administration routes, including (b) (4) iniection. After dosing, the favorable safety profile is notable because it results in a higher systemic exposure than the planned IM dosing in this trial. The findings from this study were mild and mostly related to the mode-of-action and the RNA-intrinsic stimulation of innate immune sensors. In rodents, the non-clinical safety profile of uRNA and modRNA was predictive for clinical safety.
RNA
Relevant non-clinical safety studies
URNA
(b
4
modRNA GLP toxicity studies in mouse:
• 2 studies testing modRNA formulated in LPs (b) (4
• 2 studies testing modRNA formulated in b) (4)
Non-GLP safety studies in (b) (4)
:
. 4 studies testing modRNA formulated in LNPs (b) (4)
Route: (b) (4)
(other routes in R&D studies)
Dose: up to 200 pg/mouse, up to 1.6 mg/kg in (b) (4) monkeys
Overall safety conclusions:
No test item related findings were noted for (b) (4)
formulated modRNA after(b) (4)
administration. (administration of high doses of LP formulated modRNA was generally well tolerated.
Slight to moderate effects on hematology, clinical parameters (liver enzymes) and on lymphoid organs (spleen, liver) were noted, but were ameliorated after a 2-3 wk recovery period
In studies in (b) (4) monkeys almost no test-item related findings were noted on safety parameters Slight, but reversible effects on hematology were detected, that were attributed to the mode of action of the encoded proteins.
saRNA
(b) (4)
(b) (4)
Figure 1: BioNTech non-clinical platform experience
Pre-IND meeting was held for this IND on April 06, 2020.
f

BLA 125742
Nonclinical:
Sponsor Ouestion 2:
Does CBER agree that the proposed contents of the nonclinical package, including interim results of the ongoing pivotal GL rat toxicity study (38166), will be sufficient to support initiation of the planned Phase 1/2 study in the US?
Regarding the ongoing pivotal GLP rat toxicity study (38166), the initial IND will include an interim report with the in-life endpoints (including clinical pathology and partial cytokine results) from the dosing phase. The dosing phase histology, remaining cytokine results, all serology results, and all the recovery phase endpoint results will be submitted as soon as they become available, but no later than 120 days after submission of the IND. Does CBER agree?
FDA Response to Ouestion 2:
We agree that the proposed contents of the nonclinical package, including interim results of the ongoing pivotal GLP rat toxicity study (38166), will be sufficient to support initiation of the planned Phase 1/2 study in the US. We also agree to accept an interim report of the in-life endpoints in the initial IND with the remainder being submitted at a later point in time but no later than 120 days after submission of the IND.
Proposed clinical study:
The clinical study is a multi-site, phase I/II, 2-part, dose-escalation trial investigating the safety and immunogenicity of four prophylactic SARS-CoV-2 RNA vaccines against COVID-2019 using different dosing regimens in healthy adults.
In this study four different vaccines (BNT162a1, BNT162b1, BNT162b2, and BNT162c2) will be tested. Two parts will be included in this study:
Part A
A dose-finding part with four dose cohorts (treatment groups) for each vaccine and one predefined and one optional dose level for a de-escalation approach. A dose-escalation design will be followed in the first part of the trial (part A). Subiects in this trial (first-in-human [FIHI immunization) will be immunized using a sentinel dosing/subject staggering (EMA 2017 guidance "Strategies to Identify and Mitigate Risks for First-in-Human and Early Clinical Trials with Investigational Medicinal Products"). The table below shows the FIH starting dose and the planned escalation/de-escalation doses:
7

BLA 125742
Vaccine encoded antigen
Vaccine IM dosing regimen
Part A - Dose Groups & Dose (Ng)
(12 subjects per cohort)
Vaccine
mRNA
туре
1
Starting dose
2
3
De-escalation dose
BNT162a
1
URNA
RBD of the SARS-CoV_
2 S protein
Prime:
Day 1
Boost:
Day 22
(b) (4)
BNT1626
1
modRN
A
RBD of the S protein
Prime:
Day 1
Boost:
Day 22
1B
10 pg
(b) (4)
2B
30 Mg
BNT1626
2
modRN
A
A modified
Prime:
version of
Day 1
the
Boost:
S protein
Day 22
1C
10 g
2C
30 ug
3С
1 g
BNT162c
2
saRNA
A modified version of the
S protein
Prime only:
Day 1
(b) (4)
IM = intramuscular, RBD = Receptor Binding Domain; S protein = SARS-CoV-2 Spike protein
Figure 2: Summary of vaccine dose regimens in the clinical study
PART A : Dose Cohort Scheme for uRNA (BNT162a1) and saRNA (BNT162c1)
(b) (4)
Part B - Optional
Expansion
Cohorts
4
4B
100 Ng
4C
100 g
As above
As above
As above
Figure 3: Part A; Dose cohort scheme for uRNA (BNT162a1) and saRNA (BNT162c1)
§

BLA 125742
PART A: Dose Cohort Scheme for modified RNA groups (BNT162b1 and BNT162b2)
Cohort 3
Dose level 1 ug
Cohort 1
Dose 10 ug
Cohort 2
Dose Level 30 ug
Cohort 4
Dose Level 100 g
12 subiects
Day 1: DL1 Sentinel
Group (1 subject)
Day 1: DL1 Sentinel
Group (2 subjects)
Day 1: DL1 Sentinel
Group (2 subjects)
24 h Safety Reviev
24 h Safety Reviev
24 h Safety Reviev
Day 2: 5 subjects
Day 2: 4 subiects
Day 2: 4 subjects
Based on safety data for 2 6 subjects
48 h Safety Review
Day 4: 6 subiects
Based on safety data for
₴ 6 subiects
48 h Safety Revievi
Day 4: 6 subjects
Based on safety data for
≥ 6 subiects
48 h Safety Reviev
48 h Safety Reviev
48 h Safety Review
Day 4: 6 subjects
48 h Safety Review
SRC Review
SRC Review *
SRC Review *
SRC Review *
*The data assessed by the SC for progression comprises 48 h data for 6 subjects
Figure 4: Part A; Dose cohort scheme for modified RNA groups (BNT162b1 and BNT1622)
DL = Dose level; SRC = Safety Review Committee
Figure of graphical depiction of the dose-finding process in part A
Part B
Dedicated to recruit expansion cohorts with dose levels which are selected from data generated in part A. Using a P/B regimen, the vaccines BNT162a1, BNT162b1, and BNT162b2 will be administered. For the vaccine BNT162c2, SD regimen will be used. After evaluation of aggregate data from part A, details of part B will be defined using a protocol amendment. Based on analysis of both immunogenicity and safety data gathered in part A, progression to part B will be decided. Immunogenicity and safety will be thoroughly assessed to select the vaccine and the dose(s) to be further evaluated in part B.
Safety data to be evaluated includes the package used by the SRC to assess individual dose levels. Immunogenicity of all doses will be assessed. In the protocol amendment, a summary of relevant safety and tolerability data collected in part A will be included. Also, the protocol amendment will include part B specific inclusion/exclusion criteria, obiectives/endpoints, a description of the planned statistical analyses, and descriptions of any added trial assessments and procedures.
The design of part B will be a randomized, placebo-controlled in the likely target population (e.g., high risk populations such as elderly and/or immunocompromised populations). Part B may employ a surrogate marker as a measure of vaccine efficacy.
Studies reviewed for this BLA:
1-Repeat-dose toxicity study of three LN-formulated RNA platforms encoding for viral proteins by repeated intramuscular administration to Wistar Han rats. Study number:
38166 (submitted in amendment 0).
Ç

BLA 125742
2- 17-day intramuscular toxicity study of BNT162B2 (V9) and BNT162B3C In Wistar Han rats with a 3-week recovery. Study number: 20GR142 (submitted in amendment 32).
3- A Combined Fertility and Developmental Study (Including Teratogenicity and Postnatal
Investigations) of BNT162b1, BNT162b2 and BNT162b3 by the Intramuscular Administration in the Wistar Rat. Study number: 20256434 (submitted in amendment 141).
Studies not reviewed in all amendments:
None.
Toxicology Study Review
Study number 1:
Title and study number: Repeat-dose toxicity study of three LNP-formulated RNA platforms encoding for viral proteins by repeated intramuscular administration to Wistar Han rats. Study number: 38166.
Performing laboratory: (b) (4)
Study initiation date: March 17, 2020
Final report date: July 1, 2020
Test article batch/lot:
Test Article
Buffer (b) (4)
b) (4)
(BNT162a - 1)
(b) (4)
(BNT1626 - 1)
RBP020.1" (BNT1626 - 2)
(b) (4)
(BNT162c - 1)
Batch Number (b) (4)
(b) (4)
(b) (4)
CoVVAC/160320 (b) (4)
Animal species and strain: Rat/Wistar/®):WI(Han)
Breeder/supplier: b) (4)
Stability
Not reported
Not reported
Not reported
Not reported
Not reported
Number of animal per group and sex: 15/sex/group
Age: Approximately 10-14 weeks at 1st dosing
Body weight range:
Males: 252.8g-343.9g
Females: 188.3g-267.3g
Route and site of administration: Intramuscular (IM)
Volume of iniection: 0.5 mL
Frequency of administration and study duration:
For groups 1 to 5 and 7:
On test days 1, 8 and 15; in total 3 administration days at one-week intervals per animal.
For group 6:
On test days 1 and 8; in total 2 administration days at one-week interval per animal.
10

BLA 125742
Dose: See study design
Stability: Analysis of stability, homogeneity and concentration of the test article under test conditions was not performed as part of the study. Stability studies were performed by the sponsor of the IND. At the time of submitting this study, stability studies with the first clinical trial material batch have just been started. Up to now no results are available. Stability data will be included in any upcoming amendment. The table below shows the protocol of stability study I for CTM drug substance batches:
(b) (4)
Table 1: Protocol of stability study I for CTM drug substance batches at different storage
conditions
Means of administration: Intramuscular (IM)
Report status: Interim report
Experimental design:
Animals were randomized and assigned to 7 different groups. Each group consisted of 18/sex/group. Groups 1 to 5 and 7 animals were dosed by IM on study days 1, 8, and 15. Groups
6 animals were dosed by IM on study days 1 and 8. The details of the study design are listed in the following table:
Group
Dose level [ug/animall
No. and sex of animals
Rat number
MS+RP+SA
MS
RP
SA
(Test item / Control)
0
(Buffer)
Control
10+5+3m
10+5+3 f
1_10
16-25
11-15
26-30
211-213
214-216
(b) (4)
2
(LNPuRNA RBD)
Test item 1
10+5+3m
10+5+3 f
31_40
46-55
41_45
56-60
217-219
220-222
11

BLA 125742
Group
Dose level lug/animall
No. and sex of animals
Rat number
MS+RP+ SA
MS
RP
SA
(Test item / Control)
(LNPuRNA RBD)
Test item 1
10 +5+3 m
10 +5+3 f
61-70
76-85
71-75
86-90
223-225
226-228
(LNP modRNA
RBD)
Test item 3
10 ÷ 5 + 3 m
10+5+3f
91-100
106-115
101-105
116-120
229-231
232-234
(b) (4)
5
(LNP modRNA
RBD
Test item 3
10+5+3 m
10+5+3f
121-130
136-145
131-135
146-150
235-237
238-240
6
(LNP saRNA RBD)
Test item 5
10+5+3m
10+5+3f
151-160
166-175
161-165
176-180
241-243
244-246
100
(LNP modRNA
Sp2)
Test item 4
10+5+3 m
10+5+3f
181-190
196-205
191-195
206-210
247-249
250-252
Erroneously
(b) (4)
treated
(LNPuRNARBD) 0+0+3 m
253-255
animals#.
Test item 1
m: male, f: female, MS: Main study, RP: Recovery period, SA: Satellite animals for cytokine analysis (except last group). #: Due to shortly planned dose reduction of group 3, three animals had already been dosed as originally planned with (b) (4) /animal. These three animals were replaced by 3 spare animals in group 3. The three erroneously treated animals were maintained for at least 48 hours as a non-GLP group with observations reported informally to the sponsor (body weight (test day 1 and 24 and 48 hours post injection), body temperature (24 and 48 hours post injection) and local tolerance (24 and 48 hours post injection)).
Table 2: Experimental design
Methods:
Randomization procedure: Yes
Statistical analysis plan: Yes.
The following parameters were evaluated: Clinical observations (twice daily), local tolerance [Draize scoring] (4, 24, and 48 hours after each injection), body weights (prior to injection on study days 1, 8, and 15, after treatment on study days 2, 9, and 16, and at necropsy on study days 10 or 17), food consumption (weekly), ophthalmology (before first dosing and at the end of the dosing period), body temperature (4 and 24 hours post injection on study days 1, 8, and 15), cytokines (study days 1, 8, 10, 15, and 17), clinical chemistry, hematology, coagulation, and
12

BLA 125742
acute phase proteins (study days 4, 10, and 17), urinalysis (study days 10 and 17), serology (day 10 [BNT162c1] or at day 17 after first immunization [BNT162a1, BNT162b1, and BNT162b2]).
Postmortem evaluations were performed on study days 10 (groups 6 and 7) and 17 (groups 1 to 5).
Parameters
Cageside observation
Clinical observations 2
Body weight
Food consumption
Body temperature
Ophthalmologic exam
Clinical chemistry*
Hematology* Coagulation*
Local tolerance [Draize scoring]
Serology
Cytokines
Urinalysis
Postmortem study evaluations
Frequency of Testing
Twice daily
Twice daily
Prior to injection on study days 1, 8, and 15, after treatment on study days 2, 9, and
16, and at necropsy on study days 10 or 17
Weekly
4 and 24 hours post injection on study days
1, 8, and 15
Before first dosing and at the end of the dosing period
Study days 4, 10, and 17
Study days 4, 10, and 17
Study days 4, 10, and 17
4, 24, and 48 hours after each injection Day 10 (BNT162c1) or at day 17 after first immunization (BNT162a1, BNT162b1, and BNT16262)
Study days 1, 8, 10, 15, and 17
Study days 10 and 17
Study days 10 (groups 6 and 7) and 17 (groups 1 to 5)
* Site collection of blood samples were retrobulbar venous plexus.
Day ofsampling
Test day 4:
At main study termination (on the day of dissection, i.e. on test davs 10 or 17):
Animals
Parameters
The first 5 main study animals Hematology per sex and group and all Clinical chemistry recovery animals.
Acute phase proteins
All main study animals
Hematology
Coagulation
Clinical chemistry
Acute phase proteins
Table 3: Blood sampling schedule for laboratory examinations
1 Cageside observations include mortality, morbidity, general health and signs of toxicity.
2 Clinical observations include evaluation of skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor and behavior.
13

BLA 125742
Parameter
Matrix
Total amount of sample
al-acid glycoprotein
Serum
150 uL
a2 macroglobulin
Serum
150 aL
Table 4: Acute phase proteins
Cytokines
Matrix
Total
Aliquots
amount of prepared sample
IFN-y
TNF-a
Serum
IL-1-B
IL-6
IL-10
Table 5: Cytokine analysis
150 uL
2 x 75 uL
Postmortem procedures:
Table of weighed organs
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Lungs
Lymph nodes (cervical (1), mesenteric (1))
Table 6: Weighed organs
Results:
No test article-related mortality was reported.
Aliquots prepared
2 × 75 uL
2 x 75 uL
Storage temperature
-20°C
‡ 10%
-20°C
‡ 10%
b) (4) Kit
Rat Alpha 1 Acid
Glycoprotein / AGP (b) (4)
Rat alpha 2
Macroglobulin(b) (4)
Storage temperature
-20°C
‡10%
Method
(b) (4)
Ovary (2)
Pituitary gland
Prostate
Spleen
Testicle (2)
Thymus
Thyroid (1) (including parathyroids)
14

BLA 125742
Clinical chemistry and hematology:
CLINICAL CHEMISTRY
MEASUREMENT RELATED
TO
END POINTS DIFFERENT THAN THE CONCURRENT CONTROL (LIST THE ENDPOINT STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great than 1.5 so indicated otherwise > 1.5))
NOT OF NOTE
ELECTROLYTE BALANCE
CARBOHYDRATE
METABOLISM
LIVER FUNCTION:
A) HEPATOCELLULAR
Alanine aminotransferase (ALT or
SGPT)
SD4 F 1 = 0.6 G7
B) HEPATOBILIARY
ACUTE PHASE REACTANTS
KIDNEY FUNCTION
OTHERS
(ACID/BASE BALANCE, CHOLINESTERASES, HORMONES, LIPIDS, METHEMOGLOBIN, AND PROTEINS)
Calcium, chloride, potassium, sodium, phosphorus
Glucose
Aspartate aminotransferase (AST or
SGOT)
Total bilirubin
Alkaline phosphatase (ALP)
Fibrinogen (also under coagulation)
Creatinine
Blood Urea Nitrogen (BUN)
Albumin (A)
Total protein
Carbon dioxide
Globulin
A/G ratio
Fasting triglycerides
SD4 M (b) (4)
SD4 M
SD4 M
SD4 M
SD4 M 1 = 0.3 G7
SpAr (b) (4)
SD4 F
SD4 F
SD4 F 1 = 0.3 G7
SD17F (b) (4)
Total Cholesterol
SDI7M (b) (4)
Creatine kinase (CK)
SD4 M(b) (4)
Gamma-GT
SD4 M (b) (4)
SD4 M SD4 M
SD4 M
SD4 M
SD4 M 1 = 3.4 G7
SD17M (b) (4)
SDI7 M
SD17 M
SD17 M
SD17M 1 = 3.0 G7
15

BLA 125742
CLINICAL CHEMISTRY
MEASUREMENT RELATED
TO
END POINTS DIFFERENT THAN THE CONCURRENT CONTROL (LIST THE ENDPOINT STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great than 1.5 so indicated otherwise > 1.5)) spar (b) (4)
SD4 F
SD4 F
SD4 F
SD4 F ^ = 4.6 G7
NOT OF NOTE
Lactate dehydrogenase (LDH)
SD4 F (b) (4)
Table 7: Serum chemistry results
Clinical chemistry results showed a decrease in ALT levels in group 7 females at study day 4.
Triglyceride levels were decreased in groups (b) (4)
7 males at study day 4. Triglyceride
levels were decreased in groups (b) (4)
7 females at study day 4. Triglyceride levels were
(b) (4) in group® females at study day 17. Cholesterol levels were (b) (4) in groups (b) (4) males at study day 17. Creatine kinase levels were b) (4) in group males at study day 4.
Gamma-GT levels were increased in groups (b) (4)
7 males at study day 4. Gamma-GT
levels were increased in groups b) (4)
7 males at study day 17. Gamma-GT levels were
increased in groups b) (4)
7 females at study day 4. LDH levels were (b) (4) in
group females at study day 4.
16

BLA 125742
Figure 5: Gamma-glutamyltransferase plasma activity in male rats mean values per group and
standard deviation. TD = Treatment day.
7-
Group 1: Control (b)
(b) (4)
Group 7: 100 g BNT162b2/animal
- - Level of mean of LPT's historical data
(b) (4)
(b) (4)
(b) (4)
Gamma-GT activity [U/L plasma]
1 -
0
Group: 1 2 3 4 5 6 7
Treatment (n = 10).
ТЫ Д
1 2 3 4 5 6 7
Treatment (n = 10)
TD 17 (Group 6: TD 10)
1 2 3 4 5 6 7
Recovery (n = 5),
TD 38 (Group 6: TD 31)
Figure 6: Gamma-glutamyltransferase plasma activity in female rats mean values per group and
standard deviation. TD = Treatment day.
Group 1: Control
(0) (4)
7-
(b) (4)
Group 7: 100 g BNT162b2/animal
- - Level of mean of LPT's historical data
5
(b) (4)
(b) (4)
(b) (4)
Gamma-GT activity [U/L plasma]
I
0-
Group: 1 2 3 4 5 6 7
Treatment (n = 10),
1 2 3 4 5 6 7
Treatment (n = 10)
TD 17 (Group 6: TD 10)
1 2 3 4 5 6 7
Recovery (n = 5),
TD 38 (Group 6: TD 31)
17

BLA 125742
Figure 7: Test article-related changes in plasma activity of gamma-glutamyltransferase compared to the control groun in %
(b)
(4)
In all test article-treated groups, an increase in albumin plasma levels and a decrease in globulin plasma levels, resulting in an altered albumin/globulin ratio, were reported. These changes are consistent with an acute phase response in albumin and globulin where albumin goes down and globulin goes up with inflammation, and the albumin/globulin ratio decreases. The following table lists the statistically significant changes reported in albumin and globulin levels and the alb./glob. ratio.
Parameter
Albumin
Statistically significant differences in albumin and globulin levels and the albumin/ globulin ratio compared to the control group
Group
Test item
Sex
2
3
4
5
BNT162a1
Dose [ug/animal!
(b) (4)
m
BNT162a1
BNT16261
BNT162b1
f
m
f
m
f
m
Test day
4
17
4
17
4
17
4
17
4
17
4
17
4
17
Change
[%]
(b) (4)
18

BLA 125742
Parameter
Globulin
Albumin/Globulin
Ratio
Statistically significant differences in albumin and globulin levels and the albumin/ globulin ratio compared to the control group
Group
Test item
Dose [ug/animal]
Sex
6
7
2
4
5
6
7
2
3
4
5
BNT162c1
BNT16262
BNT162al
BNT16261
BNT16261
BNT162c1
BNT162b2
BNT162a1
BNT162a1
BNT16261
BNT16261
100
(b) (4)
100
(b) (4)
f
m
f
m
f
m
f
m
m
f
m
f
m
m
f
m
f
m
m
f
m
Test dav
4
17
4
4
4
17
4
17
4
17
17
4
17
17
17
4
17
17
4
4
17
17
4
17
4
17
4
17
4
17
4
17
4
Change
[%]
(b) (4)
_9.1**
-5 9**
-12 6**
-11.0**
(b) (4)
+7.3*
+23.1**
+17.7※※
(b) (4)
19

BLA 125742
Statistically significant differences in albumin and globulin levels and the albumin/ globulin ratio compared to the control group
Parameter
Group
Test item
Dose [ug/animal]
Sex
Test day
17
f
4
17
6
BNT162c1
(b) (4)
m
4
f
4
7
BNT16262
100
m
17
f
4
17
Change
[%]
(b) (4)
15.1**
-23.6**
-15.7**
-24.4*※
m = Male
f= Female
*** Statistically significant at p = 0.01 / p = 0.05 (based on numerical data, not on percent difference).
Table 8: Differences in albumin and globulin levels and the albumin/ globulin ratio compared to the control group
HEMATOLOGY
MEASUREMENT
RELATED TO
Red blood cells
White blood cells
END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL (LIST THE ENDPOINT, STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great or less than 1.53. ie. >1.6 or < 1.6
Reticulocvtes
SD4M(b) (4)
SDA M
SD4 M
SD4 M SD4 M
SD4 M 1 = 0.3 G7
SD4 F (b) (4)
SDA F
SD4 F
SD4 R
SD4 F 1 = 0.5 G7
Monocvte count:
SD4F (b) (4)
SD4 F
SD17 F (b) (4)
SDIT F
SD17 F
Not of NOTE
Hematocrit (Hct)
Hemoglobin Conc. (Hb)
Mean Corp. Hb. (MCH)
Mean Corp. Hb. Conc. (MCHC),
Mean Corp. Volume (MCV)
Total Erythrocyte Count (RBC)
Macrophage
Leukocytes
3 With rounding up at the tenth decimal place. Therefore, 1.54 or less becomes 1.5 and is not reported and 1.55 or greater becomes 1.6 and is reported.
20

BLA 125742
HEMATOLOGY
MEASUREMENT
RELATED TO
END POINTS DIFFERENT THAN THE CONCURRENT CONTROL (LIST THE ENDPOINT, STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great or less than 1.53. ie. >1.6 or < 1.6
SD17 F (b) (4)
SD17 F * = 1.6 G7
Lymphocyte count
SD17 M (b) (4)
SD17 M
SD17 M 1 = 0.5 G7
Neutrophil count
SD4 M (0) 4)
SDAM
SDI7M (b) (4)
SD17 M
SD17 M
SD17 M 1 = 3.2 G7
SD4 F) (4)
SD4 F
SD4 F
SD4F1=23 G7
SPITE (b) (4)
SD17 F
SD17 F
SD17 F * = 7.8 G7
Eosinophils count
SD4 M (b) (4)
SD17M (b) (4)
SDI7 M
SD17 M
SD17 M 1 = 2.2 G7
SD17F (b) (4)
SDI7F
SD17F
SD17F 1 = 6.1 G7
Basophils
SD4 M(0) (4)
SDA M
SD4 M
SD4 M + = 2.5 G7
SDI7M (b) (4)
SD17 M SD17 M
SD17 M 1 = 2.5 G7
SD4 F (b) (4)
Not of NOTE
21

BLA 125742
HEMATOLOGY
MEASUREMENT
RELATED TO
Clotting potential
END POINTS DIFFERENT THAN THE CONCURRENT CONTROL (LIST THE ENDPOINT, STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great or less than 1.53, ie, ≥1.6 or < 1.6
SD4 F (b) (4)
SD4F1=1.7G7
SDIZE (b) (4)
SD17 F
SD17 F
SD17 F
SD17 F ^ = 2.1 G7
White Blood Cells (WBC)
SD17M (0) (4)
SDITM
SD17 M
SD17 M
SD17 M ^ = 2.2 G7
SD17F (b) (4)
SD17E
SD17 F
SD17 F
SD17 F * = 2.1 G7
Large Unstained Cells (LUC)
SD4 M (O) 4'
SD4 M
SD4 M
SD4 M
SD4 M * = 2.8 G7
SD17 M (0) (4)
SD17 M
SD17M
SD17 M
SD17 M 1 = 3.4 G7
SDir(b) (4)
SD4 F
SD4 F
SD4 F 1 = 4.2 G7
SPI7= (b) (4)
SD17 F
SD17 E
SD17 F 1 = 4.2 G7
Platelet count
SD17F(b) (4)
Fibrinogen
SD17M (b) (4)
SDITM
SD17 M
Not of NOTE
Activated partial-thromboplastin time clotting time
Prothrombin time
22

BLA 125742
HEMATOLOGY
MEASUREMENT
RELATED TO
END POINTS DIFFERENT THAN THE CONCURRENT CONTROL (LIST THE ENDPOINT, STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great or less than 1.53. ie. >1.6 or < 1.6
SD17M (b) (4)
SD17 M ^ = 3.1 G7
SDITE (b) (4)
SD17 F
SD17 F
SD17 F
SD17 F ^ = 2.6 G7
PCT %
SDI7 M(b) (4)
SD17 M
SD17 M 1 = 0.6 G7
SD17 F (b) (4)
SD17 F
SD17 F
SD17 F 1 = 0.6 G7
Not of NOTE
Others
Table 9: Hematological results
Day: 10 Relative to Start Date
Sex: Male
Bone marrow cytology
HGB
(mmolL)
(b
RBC
(X10E6/uL)
WBC
(x10E3/uL)
Haematological Parameters
Reti
Reti
(X10E3/uL)
PIT
(X10E3/pL)
al
HCT
1%'
Group 6 (b) (4)
animal
T. item 5
Mean
SD
N
4
Hematology results showed decrease in reticulocyte levels in groups (b) (4)
7 males at
study day 4. Reticulocyte levels were decreased in groups (b) (4)
7 females at study day
4. Reticulocytes levels were decreased after the 1st dose but recovered by the end of in-life of the toxicity study.
23

BLA 125742
Figure 8: Reticulocyte's levels
400-
blood
300-
200
100
0-
reticulocytes
1 (b) (4)
ange
• Gr.1 control
(b) (4)
Gr.7 BNT162b2 100 ug (b) (4)
d4 d17 d4 d17 d4 d17 d4 d17 d4 d17 d4 d17 d4 d10
Monocyte levels were (b) (4) in groups (b) (4) females at study day 4. Monocyte levels were increased in groups (b) (4)
7 females at study day 17. Lymphocyte levels were decreased
in groups (b) (4) 7 males at study day 17. Neutrophil levels were (b) (4) in group males at study day 4. Neutrophil levels were (b) (4) in group males at study day 4. Neutrophil levels were increased in groups (b) (4)
7 males and females at study day 17. Neutrophil levels
were increased in groups (b) (4) 7 females at study day 4. Eosinophil levels were (b) (4) in group males at study day 4. Eosinophil levels were (b) (4) in groups (b) (4) males at study day 17. Eosinophil levels were increased in groups (b) (4) 7 males at study day 17.
Eosinophil levels were increased in groups b) (4) 7 females at study day 17. Basophil levels were increased in groups b) (4) 7 males at study day 4. Basophil levels were increased in groups (b) (4)
7 males at study day 17. Basophil levels were increased in groups (b) (4)
7 females at study day 4. Basophil levels were increased in groups (b) (4)
7 females at
study day 17. WBC levels were increased in groups b) (4)
7 males and females at study
day 17. LUC levels were increased in groups (b) (4)
7 males and females at study day 4.
LUC levels were increased in groups (b) (4)
7 males and females at study day 17.
Platelet count were (b) (4) in group® females at study day 17. Fibrinogen levels were increased in groups b) (4)
7 males and females at study day 17. PCT% levels were
decreased in groups (b) (4) 7 males at study day 17. PCT% levels were decreased in groups ®
7 females at study day 17.
Grouns 2 and 3:
(b) (4)
94

BLA 125742
(b) (4)
Groups 4 and 5:
(b) (4)
25

BLA 125742
(b) (4)
BNT162c1 - Group 6 (b) (4)
26

BLA 125742
(b) (4)
BNT16262 - Group 7
Test article-related changes included decreases in the absolute and relative reticulocyte count, the number of platelets, and red cell mass, and increases in the numbers of leucocytes, neutrophils, monocytes, large unstained cells (LUC), basophils and/or the levels of fibrinogen. All changes fully reversed by the end of the recovery phase.
27

BLA 125742
Parameter
Test item-related changes in hematological and coagulation parameters, group 7
compared to the control group in %
Group 7: 100 Mg BNT162b2/animal
Males
Females
Test day 4
-74.3**
-47 7**
Reticulocytes (relative)
Reticulocytes (absolute)
-72.1**
-48.2**
Large unclassified cells
(LUC), abs.
Basophils (Baso), abs.
+295.5*※
+319.5*※
+150.0**
None
Test day 17
-9.1**
Hemoglobin (HGB)
Erythrocytes (RBC)
Hematocrit (HCT)
Leucocytes (WBC)
-12.7*※
None
-9 8**
-11.9**
-13.5**
+118.7**
-29.2**
+111.0**
-34.1**
Platelets (PLT)
Neutrophils (Neut), abs.
Eosinophils (Eos), abs.
+605.8**
+679.8**
+419.3**
+509.6**
Large unclassified cells,
(LUC) abs.
Basophils (Baso), abs.
+685.2**
+594.8**
Fibrinogen
+146.7*※
+205.2**
+105.3※
+1602**
abs. = absolute. None = No test item-related change. */** = Statistically significant at p < 0.01 / p < 0.05 (based
on numerical data, not on percent difference).
Table 13: test article-related changes in hematological and coagulation parameters for the treatment with BNT16262
Acute phase protein levels:
(b) (4) Parameters-Male
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL)
(ng/mL)
(b) (4) Parameters-Female
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL)
(ng/mL)
[al
[all
Group 1:
Control
Mean
SD
N
64658.6
6727.8
5
39774.6
3460.7
5
[a]
79798.8
17269.9
5
[all
18098.2
5486.8
5
Group 2:
[(b) (4)
Mean
SD
(b) (4)
28

BLA 125742
(b) (4) Parameters-Male
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL)
(ng/mL)
(b) (4) Parameters-Female
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL)
(ng/mL)
[all
all
all
animal
T. item 1
%Diff
Group 3:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
%Diff
(b) (4)
Group 5:
Mean
(b) (4) /
SD
animal
N
T. item 3
%Diff
Group 6:
Mean
|(b) (4) /
SD
animal
N
T. item 5
%Diff
Group 7:
Mean
446781.0**
100 kg/
SD
64502.0
animal
N
5
T. item 4
%Diff
591 0
2159010.0**
78652.0
5
5328.1
445614.0**
27975.1
5
458 4
1362630.0**
257962.6
5
7429.1
Cal - Anova & Dunnett (Log): ** = p < 0.01
Call - Anova & Dunnett (Rank): ** = p <0.01
Table 14: Acute phase protein levels, day 4 relatives to start date
At study day 4, alphal-acid glycoprotein and alpha macroglobulin levels were increased significantly (p < 0.01) in all treated male's and female's groups.
(b) (4) Parameters-Male
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL
(ng/mL)
(b) (4) Parameters-Female
Alhal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL)
(ng/mL)
[a]
Mean
¡(b) (4)
[a]
[al
Group 6:
(b) (4) / animal
T. item 5
[a] - Anova & Dunnett (Log): ** = p ≤ 0.01
Table 15: Acute phase protein levels, day 10 relatives to start date
29

BLA 125742
Group 1:
Control
(b) (4) Parameters-Male
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL
(ng/mL
(b) (4) Parameters-Female
Alphal-acid
Alpha2
Glycoprotein
Macroglob.
(ng/mL)
(ng/mL)
Mean
SD
N
a
50334.7
11962.9
10
[all
[al
52001.7
10058.1
10
[all
Group 2:
Mean
(b) (4) /
SD
animal
IT. item 1
Group 3:
(b) (4) / animal
T. item 1
Group 4:
Mean
|(b) (4) /
SD
animal
(b) (4)
N
T. item 3
%Diff
Group 5:
Mean
(b) (4) /
SD
animal
N
T. item 3
%Diff
Group 7:
Mean
1043631.5**
100 ug/
SD
80157.0
animal
N
10
T. item 4
%Diff
1973.4
826053.0**
274115 3
10
1488 5
[a] - Anova & Dunnett (Log): ** = p < 0.01
[all - Anova & Dunnett (Rank): ** = p < 0.01
Table 16: Acute phase protein levels, day 17 relatives to start date
At study days 10 and 17, alphal-acid glycoprotein levels were increased significantly (p <0.01) in all treated male's and female's groups.
30

BLA 125742
Cytokine levels:
Sex: Male
Group 1:
Control
Group 2:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
Group 1:
Control
Day 1 Relative to Start Date (PreDs)
Cytokine Levels
IFN-gamma
TNF-alpha
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
[a]
[al
[a]
Mean
SD
N
7.23
5.60
3
7 10
0.00
3
12.60
0.00
3
Mean
N
Mean
SD
N
(b) (4)
%Diff
Mean
SD
N
99.17
7 60
3
Day: 1 Relative to Start Date (6 h pa)
66.10
349 93
14.69
115.46
3
3
Group 2:
Mean
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
N
(b) (4)
T. item 3
%Diff
[al - Anova & Dunnett [al] - Anova & Dunnett(Log)
[a21 - Anova & Dunnett(Rank): n - Inappropriate for statistics
Table 17: Cytokine levels in males at study day 1 (b) (4)
IL-6
(pg/mL)
[a]
3.00
0.00
3
12.33
8.31
3
IL-10
(pg/mL)
[a]
9 90
0.00
3
212.37
116.87
3
31

BLA 125742
Sex: Male
Group 1:
Control
Group 2:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
Sex: Male
Group 1:
Control
IFN-gamma
TNF-alpha
Day 8 Relative to Start Date (PreDs)
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
Tal
[al
[al
Mean
SD
N
109.77
20.35
3
92 47
19.99
3
447.53
87.14
3
Mean
SD
N
%Diff
Mean
SD
)
(4)
%Diff
Mean
SD
N
88.43
19 95
3
56.80
20.82
3
Dav 8 Relative to Start Date (6 h pa)
269.07
111.47
3
Group 2:
Mean
(b) (4) /
SD
animal
T. item 1
N
%Diff
Group 4:
Mean
(b) (4) /
SD
animal
N
(b) (4)
T. item 3
%Diff
[al - Anova & Dunnett [al] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropriate for statistics
Table 18: Cytokine levels in males at study day 8
(b) (4)
IL-6
(pg/mL
[al
14 57
16.21
3
4 50
2.60
3
IL-10
(pg/mL)
[a]
365.60
74.22
3
220.07
106.23
3
32

BLA 125742
Sex: Male
Group 1:
Control
Group 2:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
Sex: Male
Group 1:
Control
IFN-gamma
TNF-alpha
Day 15 Relative to Start Date (PreDs)
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
[a]
[a]
Cal
Mean
SD
N
84.90
61 87
3
66.80
52.44
3
269.17
231.66
3
(b)
(4)
Mean
SD
N
125 33
24.16
3
82.30
36.60
3
Day 15 Relative to Start Date (6 h pa)
381.77
149.65
3
Group 2:
Mean
(b) (4) /
SD
animal
N
T. item 1
%Diff
Group 4:
Mean
(b) (4) /
SD
animal
N
(b) (4)
T. item 3
%Diff
[a] - Anova & Dunnett [all - Anova & Dunnett (Log)
[a21 - Anova & Dunnett (Rank): n - Inappropriate for statistics
Table 19: Cytokine levels in males at study day 15 (b) (4)
IL-6
(pg/mL)
[all
3.00
0.00
3
3.53
0.92
3
IL-10
(pg/mL)
[a]
178.57
147 46
3
238.63
102.97
3
33

BLA 125742
Sex: Male
Group 1:
Control
IFN-gamma
TNF-alpha
Cytokine Levels
IL-1beta
(pg/mL)
[al
Mean
SD
N
4.00
000
3
(pg/mL)
(pg/mL)
[al
[al
7.10
000
3
12.60
0.00
3
Group 2:
|(b) (4) /
Mean
SD
animal
T. item 1
Group 4:
Mean
(b) (4) /
SD
animal
N
T. item 3
%Diff
[al - Anova & Dunnett [al] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropriate for statistics
(4)
IL-6
(pg/mL)
[all
3.00
0.00
3
Table 20: Cytokine levels in males at study day 17 relatives to start date (48h pa)
(b) (4)
Sex: Female
Group 1:
Control
| Group 2:
ug/ animal
T. item 1
Group 4:
(ole
ug/ animal
T. item 3
Group 1:
Control
IFN-gamma
TNF-alpha
Day 1 Relative to Start Date (PreDs)
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
Mean
SD
N
[a]
30.67
46.19
3
[al]
28.57
23 95
3
[al]
119.00
135.10
3
Mean
well.
N
(b) (4)
%Diff
Mean
SD
86.50
8.29
Day: 1 Relative to Start Date (6 h pa)
65.83
345.70
29.96
188.07
IL-6
(pg/mL)
[all
3.00
0.00
3
5.77
3.19
IL-10
(pg/mL)
[al]
9.90
IL-10
(pg/mL)
[al]
71.90
107.39
3
168.03
78.07
34

BLA 125742
Sex: Female
IFN-gamma
TNF-alpha
Day 1 Relative to Start Date (PreDs)
IL-Ibeta
(pg/mL)
(pg/mL)
(pg/mL)
N
a]
3
[all
3
[all
3
Group 2:
Mean
ug/ animal
T. item 1
Group 4:
(b) (4)
ug/
'b) (4)
animal
T. item 3
N
%Diff
[a] - Anova & Dunnett [al] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropriate for statistics
Table 21: Cytokine levels in females at study day 1
Sex: Female
IFN-gamma
TNF-alpha
Day 8 Relative to Start Date (PreDs)
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
[a]
[al
[all
Group 1:
Control
Mean
SD
N
23.27
31 91
3
12.80
9 87
3
48.37
61.95
3
Group 2:
(b) (4)
ug/ animal
T. item 1
Mean
SC
N
%Diff
Group 4:
(o) (4)
ug/ animal
T. item 3
Mean
SD
(4)
%Diff
Dav 8 Relative to Start Date (6 h pa)
Group 1:
Control
Mean
SD
N
77.80
18.19
3
43.67
19.70
3
213.37
99 74
3
Group 2:
(6) (4)
ug/ animal
T item 1
Group 4:
(6) (4)
ug/ animal
Mean
(b)
N
(4)
IL-6
(pg/mL)
[al]
3
IL-6
(pg/mL)
[all
3.00
0.00
3
3.00
0.00
3
IL-10
(pg/mL)
[all
3
IL-10
(pg/mL)
[all
17.80
13.68
3
125.70
98.90
3
35

BLA 125742
T. item 3 %Diff (b) (4)
[al - Anova & Dunnett [al] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropriate for statistics
Table 22: Cytokine levels in females at study day 8
(b) (4)
Sex: Female
IFN-gamma
TNF-alpha
Dav 15 Relative to Start Date (PreDs)
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
Group 1:
Control
Mean
SD
N
[a]
37.33
57.74
3
[a]
26.27
33.20
3
[a]
116.57
180.08
3
Group 2:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
Mean
SD
N
%Diff
Mean
SD
N
(b) (4)
%Diff
Sex: Female
Group 1:
Control
Mean
SD
N
121.37
18.61
3
90.97
29.50
3
Day 15 Relative to Start Date (6 h pa)
420.53
143.71
3
Group 2:
ug/ animal
T. item 1
Group 4:
ug/
(b)
(4)
animal
T. item 3
%Diff
[al - Anova & Dunnett [al] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropriate for statistics
Table 23: Cytokine levels in females at study day 15
IL-6
(pg/mL)
[all
3.00
0.00
3
3.27
0 46
3
IL-10
(pg/mL)
[al
66.90
98.73
3
230.10
89 38
3
36

BLA 125742
(b) (4)
Sex: Female
IFN-gamma
TNF-alpha
Cytokine Levels
IL-1beta
(pg/mL)
(pg/mL)
(pg/mL)
Group 1:
Control
Mean
SD
N
[a]
32.37
49.13
3
[a]
20.03
22.40
3
[all
77.83
112.99
3
Group 2:
Mean
ug/ animal
T. item 1
Group 4:
[b) (4'
ug/ animal
N
9)
(4)
T. item 3
%Diff
[a] - Anova & Dunnett [all - Anova & Dunnett (Log)
[a21 - Anova & Dunnett (Rank): n - Inappropriate for statistics
IL-6
(pg/mL)
all
3.00
0.00
3
Table 24: Cytokine levels in females at study day 17 relatives to start date (48h pa)
(b) (4)
Urinalysis:
No test article-related effects on the urinalysis tests were reported.
Sex: Male
Group 6:
[b) (4) /
animal
T. item 5
Urinalysis pH
Specific
Gravity (g/mL)
[al
[a]
Mean
N
(b) (4)
Urine Volume
- relative - (mL/kg b.w./24 h)
[al
Table 25: Urinalysis results in males at day 10 relatives to start date
IL-10
(pg/mL)
[a]
45.87
62.30
3
37

BLA 125742
Sex: Male
Group 1:
Control
Group 2:
(b) (4) / animal
T. item 1
Group 3:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
Group 5:
(b) (4) / animal
T. item 3
Group 7:
100 ug/ animal
T. item 4
Urinalysis
Specific
pH
Gravity (g/mL)
[all
Urine Volume
- relative - (mL/kg b.w./24 h)
[all
Mean
SD
N
1.0309
0.0057
10
6 55
0.20
10
45.80
5.62
10
Mean
SD
N
Mean
SD
(b) (4)
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
1.0463 **
0.0122
10
1.5
6.35
0.27
10
-3.1
31.67**
9 65
10
-30.9
Table 26: Urinalysis results in males at day 17 relatives to start date
Specific gravity was increased significantly in groups (b) (4) 7 males at study day 17. Urine volume was decreased significantly in groups b) (4) 7 males at study day 17.
Sex: Female
Urinalvsis pH
[al
Urine Volume
- relative - (mL/kg b.w./24 h)
[al
Group 6:
(b) (4) / animal
T. item 5
Specific
Gravity (g/mL)
[a]
Mean
# (b) (4)
Table 27: Urinalysis results in females at day 10 relatives to start date
38

BLA 125742
I Sex: Female
Group 1:
Control
Group 2:
(b) (4) / animal
T. item 1
Group 3:
(b) (4) / animal
T. item 1
Group 4:
(b) (4) / animal
T. item 3
Group 5:
|(b) (4) / animal
T. item 3
Group 7:
100 ug/ animal
T. item 4
Urinalysis
pH
Specific
Gravity (g/mL)
[all
Mean
SD
N
al
1.0349
0.0047
10
6.26
0.26
10
Urine Volume
- relative - (mL/kg b.w./24 h)
al
45.54
10.71
10
Mean
SD
N
%Diff
Mean
SD
N
(b) (4)
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
1.0400
0.0099
10
0.5
6.26
0.20
10
0.0
38.35
15.62
10
-15.8
Table 28: Urinalysis results in females at day 17 relatives to start date
Specific gravity was (b) (4) significantly in group females at study day 17. Urine volume was decreased, not to significance, in groups b) (4) 7 females at study day 17.
Systemic toxicity:
No treatment-related, mortality, nor any toxicologically relevant changes in clinical signs, food consumption, body temperature, ophthalmic changes, urinalysis, or auditory examination were reported.
Treatment period for BNT162a1 - Groups 2 and 3 (b) (4)
39

BLA 125742
(b) (4)
Treatment period for BNT16261 - Groups 4 and 5 (b) (4)
Treatment period for BNT162c1 - Group 6 (b) (4)
Treatment period for BNT16262 - Group 7
On study days 1, 8, and/or 15, very slight to severe (rarely) edema were reported for all animals following the 15, 2nd d, and/or 3rd injection of 100 Mg BNT162b2/animal. All edema reported after the Is or 2nd injection had subsided by 96 hr's post administration. In addition, a few female animals revealed very slight erythema following 24 to 96 hr's following the 1st or 2nd injection.
40

BLA 125742
Skin reddening (scored as "severe" erythema) was reported in individual male and female animals at 144 hr's after the 2nd injection only but was resolved prior to the 3rd injection.
The macroscopic inspection at necropsy revealed an indurated and/or thickened injection site for 7 of 10 male and 9 of 10 female main study animals treated with 100 Mg BNT162b2/animal.
(b) (4)
Local reactions were slight after first immunization but more pronounced after boost with a reduced immunization interval.
Histopathological examination of injection sites at treatment period
Characterized mostly by moderate inflammation (up to marked) in males and moderate inflammation in females, the histopathological examination revealed test article-related injection site findings in all groups. The most severe findings were reported consistently in animals administered b) (4)
/animal and 100 g BNT162b2/animal, followed by animals
administered (b) (4)
/animal. The inflammation was characterized by infiltrates of
macrophages, granulocytes, and lymphocytes into the muscle, and variably into the dermis and subcutis. Injection site inflammation was associated with mostly moderate edema, mostly mild myofiber degeneration, occasional muscle necrosis, and mostly mild fibrosis. Skin ulceration (mild and moderate was reported in some males and females administered either b) (4)
/animal and one animal administered b) (4)
/animal. Inflammation
extended into tissues adjacent to the injection site, including mammary tissue, perineural tissue of sciatic nerve, tissue around the femur / knee and to the draining lymph node (iliac). No notable injection site findings in the control group was reported.
Body weight gain:
Test article-related treatment decreases in male's body weight gains were reported in all groups.
In females, this effect was less severe in groups (b) (4)
1. The decrease in groups (b) (4) 7
body weight gains were higher. The results of the body weight gains are reported in the figures below.
41

BLA 125742
Figure 10: Body weight gain of male rats
Figure of body weight gain of male rats treated once weekly,
mean values per group and standard deviation
Group 1: Control (b) (4)
(b) (4)
50-
40
(b) (4)
(b) (4)
Body weight gain [%]
0
10 _
Group:
^ 2
3
4 5 6 7
Treatment (n = 15),
TD 1 to TD 16 (TD 9 for group 6)
1
2 3
4 5
6 7
Recovery (n = 5),
TD 16 to TD 37 (TD 9 to TD 30 for group 6)
Figure 11: Body weight gain of female rats
Figure of body weight gain of female rats treated once weekly, mean values per group and standard deviation
Group 1: Control (b) (4)
(b) (4)
50-
Group 7: 100 ug BNT162b2/animal
Body weight gain [%]
40
30
20
(b) (4)
(b) (4)
10
0
-10-Group:
3
4 5 6 7
Treatment (n = 15),
TD 1 to TD 16 (TD 9 for group 6)
2 3 4 5 6 7
Recoverv (n = 5).
TD 16 to TD 37 (TD 9 to TD 30 for group 6)
42

BLA 125742
Organ Weight:
SEX
Males SD (10/17)
GROUPS
1
(CONTROL)
10/10
2
3
4
5
6
NUMBER OF ANIMALS
BODY WEIGHT (terminal)
BRAIN
ADRENALS-LEFT
ADRENALS-RIGHT
EPIDIDYMIDES-L
EPIDIDYMIDES-R
HEART
KIDNEYS-L
KIDNEYS-R
LIVER
LUNGS
CERV LYMPH NODES
INGUINAL LYMPH NODES
MANDIBULAR
LYMPH NODES
MESENTERIC
LYMPH NODES
POPLITEAL LYMPH NODES
PROSTATE
SPLEEN
TESTES-L
TESTES_R
PITUITARY
THYROID and
PARATHYROID
THYMUS
OVARIES
UTERUS
** =p<0.01
10/10
10/10
[a]
10/10
10/10
a]
10/10
NC/327
NC/2.00
NC/0.038
NC/0.035
NC/0 457
NC/0.419
NC/1.14
NC/1.426
NC/1.479
NC/13.02
NC/1.936
NC/0.021
4
NC/NC
NC/NC
NC/0.033
NC/NC
NC/0.927
NC/0 838
NC/1.80
NC/1.78
NC/0.013
NC/0.013
NC/0.538
NC = Not collected. L = Left; R = Right. CERV = Cervical. [a] - Anova & Dunnett: * = p < 0.05;
Table 29: Male's organ weights results. Absolute weights are expressed as mean (grams). Entries in table are expressed both as organ weight from animals taken at the end of the terminal phase and recovery phase of the study (main phase organ weight/recovery phase organ weight).
10/10
all
NC/299
NC/1.94
NC/0.043
NC/0.037
NC/0.55* NC/0.51*
NC/1.14
NC/1.390
NC/1.431
NC/12.16
NC/1.877
NC/0.016
NC/NC
NCINC
NC/0.050
NC/NC
NC/0.813
NC/1.049**
NC/1 84
NC/1_82
NC/0.011
NC/0.011
NC/0.388**
43

BLA 125742
Study day 17 male's results:
Body weight was (b) (4)
in group. Left adrenal weight was (b) (4) in groups
Right adrenal weight was increased 13% in groups (b) (4) 7. Right adrenal weight was (b) (4)
in groups (b) (4). Right adrenal weight was (b) (4)
in groups
,, respectively. Left epididymides weight was increased (b) (4) , and 20% in groups (b) (4) and 7, respectively. Right epididymides weight was increased (b) (4)
and 22% in
groups (b) (4) and 7, respectively. Liver weight was (b) (4)
"In group®. Corvical lymph
node's weight was decreased 24% in group 7. Mesenteric lymph node's weight was increased (b) (4)
and 52% in groups (b) (4) and 7, respectively. Prostate weight was decreased
(b) (4)
and 12% in group (b) (4) and 7, respectively. Spleen weight was increased (b) (4) and 25% in groups (b) (4) and 7, respectively. Thymus weight was decreased
(b) (4)
and 28% in groups (b) (4) and 7, respectively.
SEX
Females SD (10/17)
GROUPS
NUMBER OF ANIMALS
BODY WEIGHT (terminal)
BRAIN
ADRENALS-L
ADRENALS-R
EPIDIDYMIDES_I
EPIDIDYMIDES-R
HEART
KIDNEYS-L
KIDNEYS-R
LIVER
LUNGS
CERV LYMPH NODES
INGUINAL LYMPH NODES
MANDIBULAR
LYMPH NODES
MESENTERIC
LYMPH NODES
POPLITEAL
LYMPH NODES
PROSTATE
SPLEEN
TESTES-L
TESTES-R
PITUITARY
THYROID and
PARATHYROID
THYMUS
1
(CONTROL)
10/10
[a]
NC/221
2
3
4
5
6
10/10
10/10
10/10
10/10
[a]
[a]
[a]
[a]
10/10
[a]
NC/1.86
NC/0.045
NC/0.044
NC/0.914
(b) (4)
NC/0.938
NC/0 989
NC/8.35
NC/1 333
NC/0.016
NCINC
NCINC
NC/0.034
NC/NC
NC/0.595
NC/0.015
NC/0.013
NC/0.456
10/10
a
NC/219
NC/1.87
NC/0.049
NC/0.049
NC/0.866
NC/1.009
NC/1.057
NC/9.95**
NC/1.524
NC/0.017
NC/NC
NCINC
NC/0.037
NCINC
NC/0 957**
NC/0.014
NC/0.011
NC/0 390
44

BLA 125742
SEX
Females SD (10/17)
GROUPS
1
2
3
4
5
6
(CONTROL)
NUMBER OF
10/10
10/10
10/10
10/10
10/10
10/10
ANIMALS
[a]
[a]
[a]
[a]
[a]
[a]
OVARY-L
NC/0.054
OVARY_R
NC/0.058
UTERUS
NCINC
(b) (4)
NC = Not collected. L = Left, R = Right. CERV = Cervical. [a] - Anova & Dunnett: * = p <0.05;
** =p<0.01
Table 30: Female's organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed both as organ weight from animals taken at the end of the terminal phase and recovery phase of the study (main phase organ weight/recovery phase organ weight).
Study day 17 female's results:
Left and right adrenal weight was (b) (4)
in group. Left kidney weight was (b) (4)
in group w. Liver weight was increased (b) (4)
19% in groups (b) (4) 7,
respectively. Lungs weight was increased b) (4)
14% in groups (b) (4)
7, respectively. Cervical lymph node's weight was increased (b) (4)
in groups
(b) (4) , respectively. Mesenteric lymph node's weight was (b) (4) in group h
Mesenteric lymph node's weight was (b) (4)
in groups (b) (4), respectively.
Spleen weight was increased (b) (4)
61% in groups (b) (4)
7.
respectively. Thyroid weight was decreased (b) (4)
15% in groups (b) (4)
7,
respectively. Thymus weight was decreased (b) (4) 14% in groups (©) (4) 7, respectively.
Gross pathology:
Test article-related findings in all groups included injection site findings, enlarged iliac lymph nodes, and enlarged spleen. All other findings were considered incidental.
Groups Findings
1M
Emphysematous-lungs (1/10); reddened thymus (1/10)
2M
3M
4M
(b) (4)
5M
10/10
[a]
NC/0.049
NC/0.056
NC/NC
45

BLA 125742
Groups Findings
6M
(b) (4)
7M
Indurated injections site It II (5/10); enlarged iliac lymph nodes (5/10); enlarged renal lymph nodes (1/10); enlarged spleen (2/10); thickened injection sites I+II (1/10)
Table 31: Male's gross pathology results.
Groups
Findings
1F
No findings
2F
3F
4F
(b) (4)
5F
6F
7F
Indurated injections site I (3/10); indurated injections site IHII (4/10); enlarged iliac lymph nodes (7/10); enlarged spleen (7/10); thickened injection sites I (2/10); muscle jellied [adhered to sciatic nerve and bone] at injection site I (1/10); dilated uterus [filled with clear liquid] (1/10); sciatic nerve adhered to injection site I (2/10)
Table 32: Female's gross pathology results.
Microscopic findings:
Terminal sacrifice
Inflammation at the injection site and surrounding tissues, increased cellularity of germinal centers and increased plasma cells in the draining (iliac) lymph node, increased cellularity (hematopoiesis) in the bone marrow and spleen, and vacuolation of hepatocytes in the portal regions were the test article-related microscopic findings reported at the end of dosing period. At the end of the 3-week recovery phase, all microscopic findings were partially or fully recovered.
46

BLA 125742
In all groups, test article-related injection site reactions were reported. Site reactions were mostly characterized by moderate inflammation (up to marked) in males and moderate inflammation in females. In groups (5) (4) 7 ((b) (4)
100 ug BNT162b2/animal), the most
severe findings were consistently reported. Followed by the animals administered (b) (4)
/animals. The inflammation at the injection site was characterized by infiltrates of
macrophages, granulocytes, and lymphocytes into the muscle, and variably into the dermis and subcutis. Injection site inflammation was associated with mostly moderate edema, mostly mild myofiber degeneration, occasional muscle necrosis, and mostly mild fibrosis. In some males and females treated with either (b) (4)
/animal and one animal administered (b) (4)
animal, skin ulceration (mild and moderate was reported. At the end of the 3-week
recovery phase, injection site findings were partially recovered. The inflammation at the inection sites were extended into tissues adjacent to it. The adjacent tissues included mammary tissue, perineural tissue of sciatic nerve, tissue around the femur/knee and to the draining lymph node (iliac). At the end of the 3-week recovery phase, these findings were mostly recovered.
In the draining (iliac) lymph node, test article-related findings were characterized by increased cellularity of the follicular germinal centers and increased plasma cells (plasmacytosis) and were variably present in all groups. In all test article-treated groups, minimal to mild increases in the cellularity of bone marrow were reported. They were likely secondary to inflammation-related platelet activation and consumption. Also, extramedullary hematopoiesis in the spleen were reported. A test article-related vacuolation of hepatocytes in the portal regions of the liver was reported in all groups.
A few other minor microscopic changes were recorded for other organs and were not considered test article-related. All changes are regarded to be spontaneous in nature being within the normal background pathology commonly reported in rats of this strain and age.
(b) (4)
47

BLA 125742
(b) (4)
.../... number of animals affected per number of animals examined
* significantly different from control (p <0.05)
** significantly different from control (p <0.01)
Table 33: Incidences of test article-related microscopic findings for the animals treated with
BNT162al
(b) (4)
48

BLA 125742
(b) (4)
.../... number of animals affected per number of animals examined
* significantly different from control (p < 0.05)
** significantly different from control (p <0.01)
Table 34: Incidences of test article-related microscopic findings for the animals treated with BNT162b1
(b) (4)
49

BLA 125742
Incidences of test item-related microscopic findings in male and female main study animals after terminal sacrifice on test day 10 (group 6) or test day 17 (group 7)
BNT162 c1
BNT162 b2
Organ / Finding
(b) (4)
Group 7: 100 ug/animal
Males
Females
10/1038
10/10*8
(b) (4)
Perineural tissue of sciatic nerve:
- Inflammation (perineural)
Bone, os femoris with joint (surrounding tissue):
- Inflammation
Mammary gland (Interstitial tissue):
- Inflammation
Lymph node (iliac):
- Plasmacytosis
- Inflammation
- Increased cellularity, germinal center
Skeletal muscle:
- Infiltration, lymphohistiogranulocyt.
Spleen:
- Increased haematopoiesis
Liver
- Vacuolation, hepatocellular, periportal
2/10
2/10
10/10**
9/10**
10/10
5/10*
2/10
9/10**
.../... number of animals affected per number of animals examined
* significantly different from control (p < 0.05)
** significantly different from control (p <0.01)
9/10**
0/10
10/10**
6/10*
10/10**
0/10
8/10**
10/10**
Table 35: Incidences of test article-related microscopic findings for the animals treated with BNT162c1 and BNT16262
50

BLA 125742
Table 36: Microscopic findings at terminal sacrifice
Observations:Neo-PlasticananonNeo-Plastic--
--MALES..
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group7:
Number of Animals on Study:
Number of Animals Completed:
ADRENAL GLAND, LEFT;
Examined...…
Dilation vascular
86.7%
13.3%
ADRENAL GLAND, RIGHT;
Examined..............
WithinNormallimits
Dilation:vascular
(15)
86.7%
13.3%
BONE, OS FEMORIS WITH JOINT;
Examined................
WithinNormalLimits.........
Inflammation; mixed; surrounding tissue.
(15)
100.0%
0.0%
BONE MARROW, OS FEMORIS WITH JOINT;
Eyamined
WithinNormallimits……
IncreasedCellularitV…wawwwww
100.0%
0.0%
CERVIX;
Examined.. ...
WithinNermalimits
Keratinization:epithelial
(-)
EPIDIDYMIS, LEFT;
Examined.............
WithinNormallimits……
Infiltration.Lvmphocvtic.
(15)
26.7%
73.3%
EPIDIDYMIS, RIGHT;
Examined...………………....
WithinNormallimits…….....
Infiltration Ivmphorvtic
(15)
33.3%
66.7%
HEART:
Evamined
WithinNormallimits.……
Infiltration:(vmphohistiocvtic.........
Infiltration:mixed............
Infiltration, Lymphocvtic...
(15)
100.0%
0.0%
0.0%
0.0%
-FEMALES.
- .Removal Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
(b) (4) = (b) (4)
(15)
(15)
73.3%
80.0%
20.0%
20.0%
80.0%
20.0%
(15)
73.3%
20.0%
(15)
86.7%
13.3%
(15)
93.3%
6.7%
(15)
6 7%
13.3%
(15)
33 3%
66.7%
(15)
100.0%
0.0%
(15)
100.0%
0 0%
(15)
80.0%
20.0%
(-)
(15)
40.0%
60.0%
(15)
33.3%
66.7%
(14)
78.6%
21.4%
(-)
(15)
26.7%
73 3%
(15)
13.3%
86.7%
(15)
86.7%
0.0%
0.0%
13.3%
(15)
100.0%
0.0%
0.0%
0.0%
(15)
100.0%
0.0%
0.0%
0.0%

BLA 125742
Observations:Neo-PlasticandNonNeo-Plastic-
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group7:
Number of Animals on Study:
Number of Animals Completed:
INJECTION SITE I;
Within NormalLimits..................
Fibrosis: intramuscular / interstitial.
FIbrosis:inter- perimuscular….
Inflammation' vmphohistiocutic
intramuscular/interstitial....
Inflammation: vmphohistiocvtic: inter-/
Inflammation: mixed:suocutis….....
Inflammation; mixed; intramuscular /
interstitial .................
Inflammation; mixed; inter-/
perimuscular ........…..........
Degeneration: mvofiber
Edema subcutis……
Edema; intramuscular / interstitial......
Edema:inter-/perimuscular……
Herolasia: epidermal ………………
INJECTION SITE II:
Fyamined...........
WithinNormalimits.................
Degeneration:mvofiber…………
Regeneration;muscle..........
Hvernlasia:epidermal.……………
Scab;epidermal.
Edomarcubeutic
Edema inter_/nerim scular
Edema intramuscular /interstitial
Necrosis:mvoriber..
Mocrocic-dermis•subcutis...……
Necrosis:traumatic: mvofiber…
Fibrosis cubcutic
Fibrosis:inter-/perimuscular…
Fibrosis intramuscular /interstitial. 00% (b) (4)
-MALES.
-FEMALES.
(b) (4)
(15)
60.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
a (b) (4)
(657 as (b) (4)
66.7% 0.0%
0.0%
0.0%
0.0%
cos (b) (4)
(b) (4)
60.0% 0.0% (b) (4)
66.7%
66.7%
66.7%
(b) (4)
(15)
0.0%
66.7%
0.0%
46.7%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
66.7%
66.7%
66.7%
0.0%
0.0%
0.0%
6.7%
100.0%
86.7% 0.0% (b) (4)
80.0%
-Removal Reasons: All of those SELECTED
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
(15)
0.0%
93.3%
100.0%
(b) (4) =
(15)
66.7%
0.0%
0.0%
6.7%
93.3%
93.3%
20.0%
0.0%
33.3%
0.0%
66.7%
0.0%
66.7%
0.0%
66.7%
66.7%
0.0%
0.0%
0.0%
66.7%
(15)
66.7%
6.7%
6.7%
26.7%
26.7%
66.7%
66.7%
66.7%
66.7%
66.7%
(15)
6.7%
66.7%
0%
60.0%
0.0%
66.7%
66.7%
66.7%
6.7%
0.0%
52

BLA 125742
Observations:Neo-PlasticandNonNeo-Plastic-
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group7:
Number of Animals on Study:
Number of Animals Completed:
Inflammation; mixed;subcutis.
_intammation•miyer•intor.
perimuscula
Intlammation: mixed:intramuscular/
interstitial
0.%% () (4)
INTESTINE, RECTUM;
Examined.
WithinNormalLimits………………………………
Infiltration. Eosinophilic: increased..
Hyperplasia; mucosa-associatedlymphoid
KIDNEY, LEFT;
Examined.
WithinNormalLimits
Congestion ......
Rasonhilia:tubule
Infiltration, Lymphocytic...
KIDNEY, RIGHT;
Examined.
WithinNormalLimits
Congestion GOODBranDa.
Basophilia:tubule…..
Infiltration Ivmphorvtic
LIVER;
Fyamined…...…………….
WithinNormallimits.……...
Congestion
Hematonoiesis'eytramedullarv
Infiltration: mixed……
Mocracie
Infiltration Neutronhilic.....
Infiltration.Lvmphocvtic….
Vacuolation:hepatocellular…….
Vacuolation: hepatocellular: periportal.
-MALES.
- FEMALES.
Control
(b) (4)
'* (b) (4)
66.7% 0.0% (b) (4)
(b) (4)
-Removal Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
(15)
66.7%
66.7%
0.0%
0.0%
(15)
(b) (4)
(b) (4)
66.7%
100 kg/
14
(15)
66.7%
66.7%
(15)
6.7%
93.3%
13.3%
26.7%
(15)
6.7%
93.3%
0.0%
6.7%
(15)
0.0%
100.0%
13.3%
6.7%
6.7%
6.7%
60.0%
6.7%
6.7%
(15)
6.7%
93.3%
13.3%
20.0%
(15)
0.0%
100.0%
26.7%
6.7%
(15)
0.0%
100.0%
13.3%
0.0%
6.7%
0.0%
33.3%
0.0%
60.0%
(15)
0.0%
100.0%
13.3%
6.7%
(15)
0.0%
100.0%
0.0%
6.7%
(15)
0.0%
100.0%
20.0%
888998
(b) (4) =
(15)
0.0%
100.0%
0.0%
0.0%
(15)
0.0%
100.0%
0.0%
0.0%
(15)
0.0%
100.0%
33.3%
6.7%
0.0%
0.0%
13.3%
0.0%
66.7%
Observations:Neo-PlasticandNonNeo-Plastic.-.
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group7:
_MALES.
_FEMAIES.
-Removal Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
53

BLA 125742
Number of Animals on Study:
Number of Animals Completed:
LUNGS WITH BRONCHI; (continued)
Hemorrhage;acute.
Hvperolasia:bronchial-associated
vmphoidtissue….
Infiltration, Eosinophilic; perivascular
LYMPH NODE, CERVICAL;
cxamineo............
Within NormalLimits......
Histocutosis
Erythrophagocytosis
Piementation:brown: macrophage
Hemorrhage
Plasmarutosis
IncreasedCellularitv: germinalcenter
LYMPH NODE, ILIAC;
Examined...
WithinNormalLimits…...
Histocytosis
Plasmarutosis
Infiltration.Fosinophilic……
Hemorrhage:acute………………
Inflammation ............
Infiltration:macrophage..........
Increased Cellularitv-germinalcenter
NERVE, SCIATIC;
Fyamined………
WithinNormallimits…..…
Inflammation perineural....
Vacuolation
PROSTATE GLAND;
Eyamined
WithinNormallimits………
Infiltration: mixed……
Inflammation:ourulent…
Infiltration.Lymphocvtic…….
= (b) (4) = = (b) (4)
20.0%
6.7%
13.3%
6.7%
15
46.7%
20.0%
(13)
0.0%
100.0%
0.0%
0.0%
0.0%
0.0%
100.0%
(15)
0.0%
100.0%
0.0%
6.7%
0.0%
0.0%
0.0%
86.7%
33.3%
53.3%
(15)
6.7%
86.7%
0.0%
0.0%
0.0%
6.7%
93.3%
(15)
0.0%
93.3%
0.0%
0.0%
0.0%
867%
(15)
100.0%
0.0%
0.0%
(15)
80.0%
0.0%
6.7%
13.3%
93.3%
73.3%
0.0%
0.0%
60.0%
33 3%
100 0%
(15)
20.0%
80.0%
0.0%
(15)
86.7%
0.0%
0.0%
13.3%
93.3%
0.0%
0.0%
6.7%
0.0%
0.0%
46.7%
(15)
100.0%
0.0%
0.0%
(14)
0.0%
92.9%
0.0%
0.0%
0.0%
0.0%
85.7%
(14)
0.0%
92.9%
100.0%
0.0%
0.0%
42.9%
28.6%
100.0%
(15)
26.7%
73.3%
0.0%
54

BLA 125742
Observations:Neo-PlasticandNonNeo-Plastic-
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group7:
-MALES.
-FEMALES.
- Removal Reasons: All of those SELECTED
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Number of Animals on Study:
Number of Animals Completed:
SPLEEN;
Examined...…………………………....
WithinNormal imits
Congestion
Hematopoiesis;increased.
STOMACH, GLANDULAR;
Examined……
WithinNormalimits………………………
Infiltration.Eosinophilic...........
Intiltration "vmphocvtic
Dilation:alandular...…
THYMUS;
Examined….
WithinNormalLimits….
VSt ...................................
Hemorrhage;acute...
UTERUS;
WithinNormalLimits..……
Dilation ....
VAGINA:
Examined.. ...............
WithinNormalLimits......
Keratinization; epithelial....
I (b) (4) : = (b) (4)
20.0%
40.0%
80.0%
13.3%
0.0%
80.0%
0.0%
(15)
6.7%
93.3%
0.0%
0.0%
(15)
66.7%
0.0%
33.3%
(15)
0.0%
93.3%
0.0%
6.7%
(15)
6.7%
93.3%
0.0%
0.0%
15
(15)
(15)
13.3%
66.7%
53.3%
(15)
20.0%
73.3%
0.0%
13.3%
53.3%
0.0%
46.7%
(15)
46.7%
0.0%
53.3%
0.0%
60.0%
(-)
(-)
(15)
100.0%
0.0%
(15)
73.3%
26.7%
(15)
93.3%
6.7%
(15)
60.0%
40.0%
55

BLA 125742
Recovery sacrifice
At the end of the recovery period (day 31 for group; and day 38 for all other groups), most of the microscopic findings reported at the injection sites, iliac lymph node, surrounding tissue of the injection sites (surrounding tissue of bone, os femoris with joint; perineural tissue of sciatic nerve; interstitial tissue of mammary gland; skeletal muscle) and spleen were partially or completely recovered in all animals.
Some inflammatory lesions were still reported at the injection sites and the surrounding tissues in some animals. These lesions were less severe (minimal to mild).
The infiltration of macrophages in the iliac lymph nodes of recovery animals were regarded as a consequence of phagocytosis relating to the inflammatory reactions at the injection sites. Test article-related minimal to mild increases in the cellularity of bone marrow and extramedullary hematopoiesis in the spleen was fully recovered at the end of recovery phase.
Test article-related vacuolation of hepatocytes in the portal regions of the liver was fully recovered at the end of recovery phase. The incidence and the severity of the remaining findings were markedly reduced when compared to the main study animals.
Discussion synopsis
Inflammation was generally most at the end of dosing in groups (b) (4) 7. This is followed by or
/animal group. Ulceration at the injection site was present only in rats administered
(b) (4) . The inflammation was partially or fully resolved at the end of the recovery phase.
Increased cellularity of the germinal centers of the draining (iliac) lymph node and plasmacytosis were reported. This is consistent with the anticipated immune activation by the test articles.
Increases in bone marrow cellularity (increased hematopoiesis) and extramedullary hematopoiesis in the spleen were reported. This is consistent with a response to inflammation and immune responses induced by the test article.
Test article-related vacuolation of portal hepatocytes was reported in all groups. The vacuolation was unassociated with markers of hepatocyte damage (i.e. ALAT, ASAT) and has been reported in animals administered pegylated compounds only. This finding was fully reversed at the end of the recovery phase.

BLA 125742
Body temperature:
No test article-related effects on body temperature were reported.
(b) (4)
(b) (4)
Serology:
In this study the immunogenicity of the administered SARS-CoV-2-S protein targeted RNA vaccines BNT162a1, BNT162b1, BNT16262, and BNT162c1 was investigated. At study day 10, serum samples were collected from animals treated with BNT162c1 (group 6). At study day 17 serum samples were collected from animals treated with BNT162a1, BNT162b1, and BNT162b2
57

BLA 125742
(groups 2, 3, 4, 5, and 7). Antibody immune response analyzed by S1 domain and RBD subdomain specific (b) (4) as well as VS/SARS-CoV-2-S-based pseudovirus neutralization assay (PVNT).
All BNT162 vaccine candidates elicited a SARSCoV-2-S protein specific antibody response directed against the S1 domain and the RBD sub-domain. Antibody responses translated into neutralizing activity as reported in the VS/SARS-CoV-2-S pseudovirus neutralization test.
BNT162 vaccine candidates showing higher antigen-specific antibody titers also displayed more pronounced virus neutralization effect.
Figure 14: Antibody titer resulting in 50% pseudovirus neutralization activity (pVN50).
Individual VNT titers resulting in 50% pseudovirus neutralization (p VN50) are shown by dots; group mean values are indicated by horizontal bars (‡SEM, standard error of the mean).
(b) (4)
(6)
(b
@titer
serum dilution =
BNT162a1
(b)
BNT16261
BNT162c1 BNT162b2
(4)
ULOQ
LLOQ
(b) (4)
(b) (4).
(b) (4)
1004g modRNA-VOB (d17)
Figure 15: antibody titer resulting in (b) (4) pseudovirus neutralization activity (pVN50).
Individual VNT titers resulting in (b) (4) pseudovirus neutralization (pVN° ) are shown by dots; group mean values are indicated by horizontal bars (‡SEM, standard error of the mean).
(b) (4).
(6)
Eliter
(4)
[serum dilution &
(b)
(4) =
LLOQ
(b)
) (4)
(4)
100Ng modRNA-VO8 (d17)
58

BLA 125742
Test article related effects
† Triglycerides ^ Gamma-GT
1 Reticulocytes
I. Platelet
^ Monocytes
^ Neutrophils
^ Eosinophils
† Basophils ^ WBC
^ LUC
^ Fibrinogens
^ РСТ%
^ Alphal-acid glycoproteins
† Alpha2-macroglobulins
^ Epididymides weight
^ Mesenteric lymph nodes weight
^ Spleen weight
^ Thyroid weight for females
Injection site findings (indurated, incrusted, and thickened skin)
Enlarged iliac lymph nodes
Enlarged spleen
^ Cellularity of bone marrow
Immune responses in groups (b) (4) and 7
Table 37: Test article related effects
Effects considered incidental
1 Thymus weight
IFN-gamma, TNF-alpha, IL-Ibeta, and IL-10
Assessment:
No treatment-related, mortality, nor any toxicologically relevant changes in clinical signs, food consumption, body temperature, ophthalmic changes, urinalysis, or auditory examination were reported.
A triglyceride is an ester derived from glycerol and three fatty acids.+ Triglycerides are the main constituents of body fat in humans and animals, as well as vegetable fat. They are also present in the blood to enable the bidirectional transference of adipose fat and blood glucose from the liver, and are a major component of human skin oils. In the human body, high levels of triglycerides in the bloodstream have been linked to atherosclerosis and, by extension, the risk of
4 "Nomenclature of Lipids". IUPAC-IUB Commission on Biochemical Nomenclature (CBN). Retrieved
2007-03-08
5 Ne/son, D. L.; Cox, M. M. (2000). Lehninger, Principles of Biochemistry (3rd ed.). New York: Worth Publishing. ISBN 1-57259-153-6.
6 Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, M.
(1983). "Human stratum corneum lipids: characterization and regional variations". J. Lipid Res. 24: 120-
130. PMID 6833889
59

BLA 125742
heart disease and stroke.& The decrease in triglyceride levels were not considered of any toxicological importance.
Gamma-glutamyl transferase (GGT) is a membrane-bound enzyme catabolizing reduced glutathione to cysteine and glycine in Meister's y-glutamyl cycle (Orlowski and Meister, 1970).?
This delivers cysteine for intracellular synthesis of glutathione, the major thiol anti-oxidant.
Elevated serum levels of GGT are markers of oxidative stress, resulting from factors including alcohol, heavy metals, cardiovascular disease and diabetes. Furthermore, higher serum levels of GGT, within the normal range, are associated with an increased cancer risk. High levels of GGT seem to increase the risk of progression of high-grade cervical dysplasia to invasive carcinoma. 10
Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow and then circulate for about a day in the blood stream before developing into mature red blood cells. Like mature red blood cells, in mammals, reticulocytes do not have a cell nucleus. I Abnormally low numbers of reticulocytes can be attributed to chemotherapy, aplastic anemia, pernicious anemia, bone marrow malignancies, problems of erythropoietin production, various vitamin or mineral deficiencies (iron, vitamin B12, folic acid), disease states (anemia of chronic disease) and other causes of anemia due to poor RBC production. 12
The cells that circulate within our blood and bind together when they recognize damaged blood vessels are called platelets. The platelets bind to the site of the damaged vessel in any cut, thereby causing a blood clot to stop bleeding. Platelets are literally shaped like small plates in their non-active form. A damaged blood vessel will send out a signal and when platelets receive that signal, they'll respond by traveling to that area and transform into their "active" formation.
To make contact with the broken blood vessel, platelets grow long tentacles and then resemble a spider or an octopus. A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. Having more than 450,000 platelets is a condition called thrombocytosis; having less than 150,000 is known as thrombocytopenia. A decrease in platelet levels is called thrombocytopenia. Easy bruising, and frequent bleeding from the gums, nose, or GI tract are the symptoms of thrombocytopenia. Thrombocytopenia happens when something is preventing your body from producing platelets. There are a wide range of causes, including: medications, an inherited condition, certain types of cancer (such as leukemia or lymphoma), chemotherapy treatment for cancer, kidney infection or dysfunction, or too much alcohol. 13
7 "Boston scientists say triglycerides play key role in heart health". The Boston Globe. Retrieved 2014-06-
18.
8 Drummond, K. E.; Brefere, L. M. (2014). Nutrition for Foodservice and Culinary Professionals (8th ed.).
John Wiley & Sons. ISBN 978-0-470-05242-6.
° Orlowski M, Meister A. The y-glutamyl cycle: a possible transport system for amino acids. PNAS. 1970;67:1248-1255.
10 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3341856/ I https://en.wikipedia.org/wiki/Reticulocyte
12 https://www.uofmhealth.org/health-library/hw203366
13 https://www hopkinsmedicine.org/heart vascular institute/centers excellence/women cardiovascular health cent er/patient information/health topics/platelets.html
60

BLA 125742
Monocytosis could be indicative of the intended immune response or could be secondary to muscle damage at the site of injection as an indication of inflammation and repair. The increases in the monocyte count might be related to test article treatment.
Neutrophils are key components in the system of defense against infection. An individual with absence or scarcity of neutrophils (neutropenia) is vulnerable to infection. The increase in neutrophils might be related to the immune responses initiated by the test article treatment.
Eosinophils are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood.
Basophils play a role in both parasitic infections and allergies. Basopenia has been reported in association with autoimmune urticaria.
White blood cells (WBCs) (also called leukocytes or leucocytes) are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic system. I4 The increase in WBC might be related to the immune response induced by the test article treatment.
LUC is a measurement of the large, peroxidase-negative cells which cannot be further characterized (i.e. as large lymphocytes, virocytes, or stem cells) present in a biological specimen. In LUC are found large lymphoid cells, more immature lymphocytes and other cells.
If the value is higher than normal, blood counts should be checked under a microscope slide.
The increases in fibrinogen levels were not considered frank toxicity but rather an anticipated effect associated with an immunological response.
Relative volume of thrombocytes (very large cells in the bone marrow called megakaryocytes)/Plateletcrit (measure of total platelet mass) percent (PCT%) was decreased in groups 3, 5, and 7 males and females at study day 17. This is crucial to normal blood clotting.
Alpha-1-acid glycoprotein (alAGp,' AGP or AAG), which is modulated by two polymorphic genes, is an acute phase (acute phase protein) plasma alpha-globulin glycoprotein. It has a normal plasma concentration between 0.6-1.2 mg/mL (1-3% plasma protein) and is synthesized primarily in hepatocytes (5). Plasma levels are affected by pregnancy, burns, certain drugs, and certain diseases, particularly HIV (5). The function of alpha-1-acid glycoprotein is to act as a carrier of basic and neutrally charged lipophilic compounds. It is known as the primary carrier of basic (positively charged) drugs (whereas albumin carries acidic (negatively charged) and neutral drugs), steroids, and protease inhibitors (5, 6). AGP shows a complex interaction with thyroid homeostasis. Alpha-1-acid glycoprotein (in low concentrations) was reported to stimulate the
14 Maton, D., Hopkins, J., McLaughlin, Ch. W., Johnson, S., Warner, M. Q., LaHart, D., & Wright, J. D., Deep V.
Kulkarni (1997). Human Biology and Health. Englewood Cliffs, New Jersey, US: Prentice Hall. ISBN 0-13-981176-1.
61

BLA 125742
thyrotropin (TSH) receptor and intracellular accumulation of cyclic AMP. However, high AGP concentrations inhibited TSH signaling (7, 8). Alpha-1-acid glycoprotein has been identified as one of four potentially useful circulating biomarkers for estimating the five-year risk of all-cause mortality (the other three are albumin, very low-density lipoprotein particle size, and citrate) (9).
Alpha-1-acid glycoprotein increases in obstructive jaundices while diminishes in hepatocellular jaundice and in intestinal infections. IS
Alpha-2-macroglobulin (a2M) is a large plasma protein found in the blood, mainly produced by the liver, and also locally synthesized by macrophages, fibroblasts, and adrenocortical cells. It acts as an antiprotease and is able to inactivate an enormous variety of proteinases. It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein and as an inhibitor of coagulation by inhibiting thrombin. Because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, TGF-B, insulin, and IL-1B, it may act as a carrier protein. In the nephrotic syndrome when other lower molecular weight proteins are lost in the urine, the concentration of alpha-2-macroglobulin rises 10-fold or more16
The epididymis is a tube that connects a testicle to a vas deferens in the male reproductive system. It is present in all male reptiles, birds, and mammals. It is a single, narrow, tightly-coiled tube connecting the efferent ducts from the rear of each testicle to its vas deferens. An inflammation of the epididymis is called epididymitis. It is much more common than testicular inflammation, termed orchitis. 17
The increases in the weights of mesenteric lymph nodes and the enlargement of the iliac lymph nodes might be related to the immune response due to test article treatment.
The external iliac lymph nodes are eight to ten in number, that lie along the external iliac vessels.
They are arranged in three groups, one on the lateral, another on the medial, and a third on the anterior aspect of the vessels; the third group is, however, sometimes absent. Their principal afferents are derived from the inguinal lymph nodes, the deep lymphatics of the abdominal wall below the umbilicus and of the adductor region of the thigh, and the lymphatics from the glans penis, glans clitoris, the membranous urethra, the prostate, the fundus of the urinary bladder, the cervix uteri, and upper part of the vagina 18
Spleen weight increase might be related to the intended immune response. The spleen plays important roles in regard to red blood cells and the immune svstem 19
. It removes old red blood
cells and holds a reserve of blood in case of hemorrhagic shock while also recycling iron. As a part of the mononuclear phagocyte system, it metabolizes hemoglobin removed from senescent erythrocytes. The globin portion of hemoglobin is degraded to its constitutive amino acids, and the heme portion is metabolized to bilirubin, which is subsequently shuttled to the liver for
15 https://en.wikipedia.org/wiki/Orosomucoid
16 https://en.wikipedia.org/wiki/Alpha-2-Macroglobulin
17 httos://en.wikipedia.org/wiki/Epididymis
18 httos://en.wikipedia.org/wiki/External iliac lymph nodes
19 Spleen, Internet Encyclopedia of Science.
62

BLA 125742
removal2. It synthesizes antibodies in its white pulp and removes antibody-coated bacteria along with antibody-coated blood cells by way of blood and lymph node circulation.
The thyroid gland controls how quickly the body makes proteins and uses energy. And, controls how sensitive the body is to other hormones. It produces the thyroid hormones [triiodothyronine (T3) and thyroxine (sometimes referred to as tetraiodothyronine (T4)]. These hormones regulate the growth and rate of function of many other systems in the body. Te and T4 are synthesized from iodine and tyrosine. The thyroid also produces calcitonin, which plays a role in calcium homeostasis. Hormonal output from the thyroid is regulated by thyroid-stimulating hormone (TSH) produced by the anterior pituitary. TSH is regulated by thyrotropin-releasing hormone (TRH) produced by the hypothalamus.
Test article-related injection site findings (indurated, incrusted, and thickened skin) were reported. Inflammation is a relatively common occurrence as part of the acute phase response following administration of some vaccines.
In all test article-treated groups, minimal to mild increases in the cellularity of bone marrow were reported. They were likely secondary to inflammation-related platelet activation and consumption.
Test article-related immune responses in groups 2, 3, 4, 5, and 7 were reported.
The thymus is a specialized primary lymphoid organ of the immune system. Within the thymus, T cells or T lymphocytes mature. T cells are critical to the adaptive immune system, where the body adapts specifically to foreign invaders. The thymus is composed of two identical lobes and is located anatomically in the anterior superior mediastinum, in front of the heart and behind the sternum.21 One of the major characteristics of vertebrate immunology is thymic involution, the shrinking of the thymus with age, resulting in changes in the architecture of the thymus and a decrease in tissue mass.22 T-cells are named for the thymus where T-lymphocytes migrate from the bone marrow to mature. Its regression has been linked to the reduction in immunosurveillance in the elderly.23
No clear important changes in the levels of cytokines (IFN-gamma, TNF-alpha, IL- Ibeta, and IL-10) were reported.
Adverse gross alteration that could be indicative of systemic or local toxicity was not reported.
Based on the overall findings in this study, it can be concluded that in Wistar rats, repeat dose on study days 1, 8, and 15 had no adverse effects in terms of systemic toxicity at the dose level of
20 Mebius RE, Kraal G. (2005). Structure and function of the spleen. Nat Rev Immunol. 5(8):606-16.
21 https://en.wikipedia.org/wiki/Thymus.
22 Shanley D.P.; Danielle A. W.; Manley N.R.; Palmer D.B.; et al. (2009). "An evolutionary perspective on the mechanisms of immunosenescence". Trends Immunol. 30 (7): 374-381. doi: 10.1016/j.it.2009.05.001
PMID 19541538
23 Linton P.J.: Dorshkind K. (2004). "Age-related changes in lymphocyte development and function". Nat. Immunol.
5 (2): 133-139. doi: 10.1038/ni1033. PMID 14749784
63

BLA 125742
10, 30, or 100 pg/animal. However, due to the significant decrease in the reticulocyte levels, hematology results should be closely monitored during any clinical trial.
LP study deviations or amendments: Deviations or amendments were not included in this study submission and expected to be included in the final study report.
Investigators Brochure: Having read and evaluated the Investigators Brochure, is it a fair, objective and reasonable summary of the toxicology data - yes (X) or no 0.
Internal Communication:
Due to the significant decreases in the platelet's and reticulocyte's levels, close monitoring to the hematology data in any clinical trial is highly recommended.
Conclusions:
Based on nonclinical toxicity assessments, there are no significant safety issues to preclude the IND from going into effect
Study number 2:
Title and study number: 17-day intramuscular toxicity study of BNT162B2 (V9) and
BNT162B3C In Wistar Han rats with a 3-week recovery. Study number: 20GR142.
Performing laboratory: Pfizer Worldwide Research & Development Drug Safety Research & Development Eastern Point Road Groton, CT 06340 USA.
Study initiation date: June 23, 2020
Final report date: August 13, 2020
Test article batch/lot:
Test Article
BNT16262 (V9)
BNT162b3c
0.9% sterile saline
Lot Number
COVVAC/270320 (b) (4)
(b) (4)
Expiration Date
27 Sep 2020
04 Dec 2020
31 Mar 2021
Animal species and strain: Rat/Wistar/).:WI(Han)
Breeder/supplier: b) (4)
Number of animal per group and sex: 15/sex/group
Age: 9 weeks
Body weight range:
Males: 243.1 grams - 291.6 grams
Females: 172.9 grams - 209.5 grams
Route and site of administration: Intramuscular (IM)
Volume of iniection: 60 ul
Frequency of administration and study duration:
Animals were treated on study days 1, 8, and 15 into the left hindlimb quadriceps muscle
Dose: See study design
64

BLA 125742
Stability: Analysis of stability, homogeneity and concentration of the test article under test conditions was not performed as part of the study. Stability studies were performed by the sponsor of the IND. At the time of submitting this study, stability studies with the first clinical trial material batch have just been started. Up to now no results are available. Stability data will be included in any upcoming amendment. The table below shows the protocol of stability study I for CTM drug substance batches:
Table of protocol of stability study I for CTM drug substance batches at different storage conditions
(b) (4)
Table 38: Protocol of stability study I for CTM drug substance batches at different storage conditions
Stability of b) (4)
was reported.
Means of administration: Intramuscular (IM)
Report status: Final report
Experimental design:
Animals were randomized and assigned to 3 different groups. Each group consisted of 15/sex/group. The first 10 animals/sex/group, by ascending animal order, were designated for necropsy at the end of the dosing phase. The remaining 5 animals were retained for the recovery phase. Animals were dosed by IM on study days 1, 8, and 15. The details of the study design are listed in the following table:
Table of experimental design
Group
Test Article or Vehicle
Number
Dose (ug RNA/Dose Day)
Dose Volume (uL/iniection site)a
60
60
(b) (4
3
30C
(b) (4)
2. Each animal received a single intramuscular injection on each dose day.
Animal Numbers
Males
Females
1-15
46-60
16-30
61-75
31-45
76-90
65

BLA 125742
b.
Sterile saline.
C.
BNT162b2 (V9).
d. BNT16263c.
Methods:
Randomization procedure: Yes
Statistical analysis plan: Yes.
The following parameters were evaluated:
General (Cageside) Clinical Days of Study
Observations:
Prior to the Initiation of Dosing
Time Points
Once daily
(PID)
Non-dosing Days (Dosing Phase)
Twice daily, except on days when detailed clinical observations were performed, then I onlv once dailv
Dosing Days (Dosing Phase)
Pre dose, except on days that pre dose detailed clinical observations were performed, 4 hours after the last animal was dosed, and at the end of the workday.
On 06 Jul 2020 (day 1), clinical signs were not conducted at the end of the workday for Animals 001-090.
Recovery Phase Davs
Twice daily
Detailed Clinical
Observations:
Detailed clinical observations were performed twice prior to the initiation of dosing, twice weekly at approximately the same time body weights were performed, and on the days) of necropsy.
Body Weight:
All animals were weighed twice prior to the initiation of dosing on PID Phase days 1 and 6, pre dose on dosing phase days 1, 8, and 15; on dosing phase days 4 and 11 (non-dosing), and a fasted weight was collected just prior to scheduled necropsy. Body weights were collected on recovery phase days 1, 4, 8, 11, 15, 18, and 21
Food Consumption:
Quantitative food consumption was recorded on dosing phase days 4, 8, 11, and 15 and on recovery phase days 4, 8, 11, 15, 18, and 21
Ophthalmology:
Ophthalmic examinations were performed once prior to the initiation of dosing (following randomization) on PID phase days 7/8 (males/females) and on dosing phase days 15/16 (males/females).
Recovery animals were not examined at the end of the recovery phase.
See the ophthalmology report in Appendix B for complete materials and methods
66

BLA 125742
General (Cageside Clinical Days of Study
Observations:
Prior to the Initiation of Dosing (PID)
Time Points
Once daily
Injection Site Scoring (Dermal Assessment):
Non-dosing Days (Dosing Phase) Twice daily, except on days when detailed
clinical observations were performed, then only once daily
Dosing Days (Dosing Phase)
Pre dose, except on days that pre dose detailed clinical observations were performed, 4 hours after the last animal was dosed, and at the end of the workday.
On 06 Jul 2020 (day 1), clinical signs were not conducted at the end of the workday for Animals 001-090.
Recovery Phase Days
Twice daily
Injection sites were observed during the dosing phase once pre dose and approximately 4 and 24 hours post dose on all animals. Animals with a score of 2 or greater at 24 hours post dose had additional evaluations at 48- and 72-hours post-dose. Animals with a continued score of 2 or greater at 72 hours post-dose had additional evaluations at 120 and 144 hours post-dose. After dosing on day 15, a 72-hour post dose evaluation was conducted on recovery animals only. Injection site score was recorded according to a standardized rating scale (Draize, 195924
Body Temperature:
On dosing phase day 1 (06 Jul 2020), pre dose dermal assessments were collected on all animals for right-side injection sites (non-injection site), and at 4 hours post dose, dermal assessments were collected on animals 1-7, 9 (group 1, males), and 46-58 (group 1, females) for right-side injection sites (non-iniection site).
Body temperature was collected on all animals once prior to the initiation of dosing on PID phase day 6, pre-dose on dosing phase days 1, 8, and 15, and at approximately 4- and 24-hours post-dose from all animals.
Table 39: parameters evaluated
Clinical laboratory measurements
Schedule for Collection of Samples for Clinical Laboratory Measurements
Parameter
Dav of Study
Dosing Phase
Recovery Phase
Hematology
Coagulation
Clinical Chemistry
Day
4
ya,c
NA
Xb,c
(Core Chemistry)
Clinical Chemistry
Xb,c
(Other Biomarkers - Acute
Phase Proteins)/Serumd
Urinalysis
NA
NA = Not applicable; X = Scheduled collection.
Day
17
Xc
XC
XC
Day
22
ус ус
XC
X
XC
X
24 Draize SH. 1959 (2nd printing 1965). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics.
Dermal Toxicity, pp. 46-59. Published by: The Association of Food and Drug Officials of the United States, Topeka, Kansas.
67

BLA 125742
a. b.
C. d. e.
First 7 animals/sex/group.
Last 8 animals/sex/group.
Blood samples were collected from animals in a fasted state, with the exception of same day redraws.
Assay performed using shared clinical chemistry sample.
Evaluated on animals scheduled for necropsy.
Table 40: Clinical laboratory measurements
Antibody (Serology) response to vaccine components
Sample Collection and Storage Conditions
Groups:
1-3
Collection Intervals:
PID Phase Day 8 and Dosing Phase Day 17ª, and Recovery Phase Day 21ª
Collection Time Points:
PID Phase Day 8, Dosing Phase Day 17, and Recovery Phase Day 21: Once
Animals/Time Point:
All animals
Anticoagulant:
No Anticoagulant
Collection Volume per
PID Phase Day 8: Approximately 0.7 mL
Sample:
Dosing Phase Day 17 and Recovery Phase Day 21: Approximately 1 mL
Sample Processing:
Samples were processed and stored as appropriate within 2 hours of collection
Sample Storage Conditions:
Approximately -60°C or lower
PID = Prior to initiation of dosing.
a. Samples collected prior to necropsy.
Table 41: Antibody (Serology) response to vaccine components
Postmortem procedures:
Animals (10/sex/group) were euthanized on dosing phase day 17 (2 days after the last dose).
Remaining animals were euthanized on recovery phase day 22.
Necropsy, tissue collection, organ weights, macroscopic tissue evaluation, and microscopic examination were performed. Bone marrow smears were collected from all animals.
Tissues Collected
Tissues Processed for Slide Preparation (X)
Organs
Weighed (All Dose
Groups)
Dose Group
Group 2
Artery, Aorta Bone Marrow, Sternum Bone, Sternum Brain Cervix Epididymis Esophagus Eye Gland. Adrenal Gland, Harderian Gland, Lacrimal (Extraorbital)
Gland, Mammary Gland, Parathyroid Gland. Pituitarv Gland, Prostate Gland, Salivary Gland, Seminal Vesicle
X
X
X
Group 1
X
X
X
x
x
X
X
X
X
X
X
X
※
x
x
X
x
XXX
X
Group 3
X
X
x
x
X
X
x
X
X
x
X
X
X
x
x
x
x
X
X
x
xXXX
X
x
x
x
x
X
68

BLA 125742
Tissues Collected
Gland, Thyroid Gut-Associated Lymphoid Tissue Heart Joint Kidney
Large Intestine, Cecum Large Intestine, Colon Larynx Liver
Lung
Lymph Node, Draining Lymph Node, Inguinal Lymph Node, Mesenteric Macroscopic Findings Muscle, Skeletal Nerve, Optic Nerve, Peripheral Ovary Oviduct Pancreas Site, Injection Skin Small Intestine, Duodenum
Small Intestine, Ileum Small Intestine, Jejunum Spinal Cord Spleen Stomach Testis Thymus Tongue Trachea Ureter Urinary Bladder Uterus Vagina
phase
Organs
Weighed (All Dose
Groups)
X
X
X
X
X
>
Tissues Processed for Slide Preparation (X)
Group 1
X
X
Dose Group
Group 2
X
X
Group 3
X
X
X
X
X
x
X
X
メメメ
x
X
X
X
X
x
X
X
X
X
X
x
X
X
x
x
X
××××
X
X
x
x
x
X
x
x
X
X
X
x
X
X
※
X
X
x
X
x
x
x
X
X
x
X
X
X
X
X
x
X
X
x
x
X
X
X
X
X
X
x
X
X
xx
X
X
X
X
X
x
X
x
x
X
X
x
x
X
X
X
X
Table 42: Tissue collection, organ weights and tissues processed for slide preparation - Dosing
Tissues Collected
Organs
Weighed (AIl Dose
Groups)
Tissues Processed for Slide Preparation (X)
Group 1
Dose Group
Group 2
Group 3
Artery, Aorta Bone Marrow, Sternum Bone, Sternum
X
X
X
69

BLA 125742
Tissues Collected
Brain
Cervix
Epididymis
Esophagus
Eye
Gland, Adrenal Gland. Harderian Gland, Lacrimal (Extraorbital)
Gland, Mammary Gland, Parathyroid Gland, Pituitary Gland, Prostate Gland. Salivary Gland, Seminal Vesicle Gland, Thyroid Gut-Associated Lymphoid Tissue Heart Joint Kidney
Large Intestine, Cecum Large Intestine, Colon Larvnx Liver Lung
Lymph Node, Draining Lymph Node, Inguinal Lymph Node. Mesenteric Macroscopic Findings Muscle, Skeletal Nerve, Optic Nerve, Peripheral
Ovary
Oviduct
Pancreas
Site, Injection Skin Small Intestine, Duodenum
Small Intestine. Ileum Small Intestine, Jejunum Spinal Cord Spleen Stomach Testis Thymus Tongue Trachea
Organs
Weighed (All Dose
Groups)
Х
X
X
Tissues Processed for Slide Preparation (X)
Group 1
Dose Group
Group 2
Group 3
X
x
X
X
X
X
X
X
X
x
x
x
X
x
x
※
x
X
X
X
X
X
※
X
X
X
X
X
X
Х
70

BLA 125742
Tissues Collected
Organs
Weighed (All Dose
Groups)
Tissues Processed for Slide Preparation (X)
Group 1
Dose Group
Group 2
Group 3
Ureter
Urinary Bladder
Uterus
Vagina
phase
Table 43: Tissue collection, organ weights and tissues processed for slide preparation - Recovery
Results:
No test article-related mortality was reported.
Clinical chemistry and hematology:
Clinical chemistry
CLINICAL CHEMISTRY
MEASUREMENT RELATED
TO
END POINTS DIFFERENT THAN THE CONCURRENT CONTROL
(LIST THE ENDPOINT STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great than 1.5 so indicated otherwise ≥ 1.5))
NOT OF NOTE
ELECTROLYTE BALANCE
CARBOHYDRATE
METABOLISM
LIVER FUNCTION:
A) HEPATOCELLULAR
B) HEPATOBILIARY
ACUTE PHASE REACTANTS
KIDNEY FUNCTION
OTHERS
(ACID/BASE BALANCE, CHOLINESTERASES, HORMONES, LIPIDS, METHEMOGLOBIN, AND PROTEINS)
Alkaline phosphatase (ALP)
SD17 F(b) (4) G3
Fibrinogen (also under coagulation)**
Albumin (A)*
GLOB*
AIG ratio*
A1A GP*
A2M*
* See table below. ** See table on page 16
Calcium, chloride, potassium, sodium, phosphorus
Glucose
Aspartate aminotransferase (AST or
SGOT)
Alanine aminotransferase (ALT or
SGPT)
Total bilirubin
Creatinine
Blood Urea Nitrogen (BUN)
Total protein
Carbon dioxide
Globulin
Fasting triglycerides
Total Cholesterol
Creatine kinase (CK)
Gamma-GT
Lactate dehydrogenase (LDH)
Table 44: Serum chemistry results for males and females
Clinical chemistry results showed an (b) (4) in ALP levels in group 3 females at study day 17.
71

BLA 125742
Dosing phase
In groups (b) (4)
, higher mean alpha-1 acid glycoprotein (A1AGP) and
alpha-2-macroglobulin (A2M) and lower Albumin:Globulin (AG) ratios (primarily due to lower albumin with slight contribution from higher globulins) on study days 4 and 17 were reported.
Dose (ug RNA/Dose Day)
Parameter
Test Article
Males
Vehicle BNT16262(V9) BNT16263c
0
30
4.
Vehicle
0
Females
BNT16262(V9) BNT16263c
30
[b) (4)
ALB (g/dL)
4D
17D
3.98
3.50
0.93x
4.16
3.60
0.86x
0.85x
GLOB (g/dL)
4D
17D
2.13
1.89
1.10x
2.10
1.84
-
1.04x
AG
4D
17D
1.88
1.85
0.90x
0.89x
1.98
1.96
0.86x
0.82x
ALAGP
4D
17D
174.358
47.672
9.42x
38.51x
239.774
95.959
7.95x
15.55x
A2M
4D
113 4
20.44x
212.1
3.32x
17D
14 0
70.76x
33.1
15.74x
Control mean values and the ratio of the test article-related findings relative to control means are listed.
-= Not test article related; A1AGP = alpha-1 acid glycoprotein; A2M = alpha-2-macroglobulin;
AG = Albumin/globulin ratio; ALB = Albumin; D = Day; GLOB = Globulin; TP = Protein, total.
Table 45: Test article-related clinical chemistry parameter effects (mean control values and ratio relative to control mean)
Recover phase
At study 22 (recovery), all test article related changes were fully reversed, with the exception of higher globulins in group (0) (4)
, and lower AG ratio in group in
Parameter
Test Article
Dose (Ng RNA/Dose Day)
Males
Vehicle BNT16262(V9) BNT16263c
0
30
(b) (4)
Vehicle
O
Females
BNT16262(V9) BNT16263c
30
(b) (4)
GLOB (g/dL)
R22
2.10
1.08x
2.26
1.06x
AG
R22
1.76
1.90
0.91x
Control mean values and the ratio of the test article-related findings relative to control means are listed.
- = Not test article related; AG = Albumin/globulin ratio; GLOB = Globulin; R = Recovery day.
Table 46: Test article-related clinical chemistry parameter effects (mean control values and ratio relative to control mean)
72

BLA 125742
Other statistically significant or apparent differences between test article and control group clinical chemistry parameters were not test article related due small magnitude of the difference and general overlap in magnitude of individual values with controls.
Hematology
HEMATOLOGY
MEASUREMENT
RELATED TO
Not of NOTE
Red blood cells
White blood cells
Clotting potential
END POINTS DIFFERENT THAN
THE CONCURRENT CONTROI (LIST THE ENDPOINT, STUDY DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great or less than 1.525, ie, ≥ 1.6 or < 1.6
HCT (%)*
Mean Corp. Hb. (MCH)*
Mean Corp. Hb. Conc. (MCHC)* Mean Corp. Hb. Conc. (MCHC)* RDW%*
Reticulocyte*
Lymphocyte count
SD17 F 1 = 1.7 G2
SD17 (b) (4)
WBC*
Neutrophil* Monocyte*
Eosinophil*
Basophil*
LUC*
Fibrinogen*
Hemoglobin Conc. (Hb)
Mean Corp. Volume (MCV)
Total Erythrocyte Count (RBC)
Macrophage
Leukocytes
Activated partial-thromboplastin time clotting time
Prothrombin time
Platelet count
Bone marrow cytology
Others
* See table on page 16
Table 47: Hematology results for males and females
Terminal phase
Hematology results showed an increase in lymphocyte levels in (b) (4)
day 17.
at study
Test article-related hematology and coagulation findings were similar in (b) (4)
However, higher mean white blood cell (WBC counts and fibrinogen concentrations, lower (day
4) and higher (day 17) reticulocyte counts, and lower red blood cell mass (red blood cell count, hemoglobin and hematocrit) were reported in b) (4)
when compared to group 1. Higher
WBC primarily involved higher neutrophils, monocytes and large unstained cells. Higher
25 With rounding up at the tenth decimal place. Therefore, 1.54 or less becomes 1.5 and is not reported and 1.55 or greater becomes 1.6 and is reported.
73

BLA 125742
eosinophils and basophils were also reported. They were present on days 4 and 17, with higher counts on day 17 than day 4. On study day 17, there were also test article-related higher fibrinogen concentrations in both sexes. Hyper-segmented neutrophils were present on peripheral blood smears of test article-treated animals.
In addition, there were test article-related transiently lower reticulocyte counts on study day 4, and higher reticuloctes on study day 17 (females only). These changes were with attendant expected changes in RBC indices (higher mean cell hemoglobin concentration; males on day 4; lower mean cell hemoglobin [MCH] and higher red cell distribution width on day 17; both sexes). These were associated with lower BC mass on davs 4 and 17 (comparable on both davs or slightly lower on day 17). Test article-related clinical chemistry findings were similar in groups 2 and 3. However, higher mean alpha-1 acid glycoprotein and alpha-2-macroglobulin and lower AG ratios (primarily due to lower albumin with slight contribution from higher globulins) were reported in (b) (4)
on days 4 and 17.
Recovery phase
After a 3-weeks recovery phase, all test article-related hematology and coagulation changes were fully reversed, with the exception of higher red cell distribution width.
There were no test article-related findings reported in urinalysis parameters in the dosing or recovery phase.
Parameter
Test Article
HCT (%)
4D
17D
MCH (pg)
17D
MCHC (g/dL)
4D
17D
RDW (%)
4D
17D
RETIC
(10e3/uL)
17D
WBC
(10e3/uL.)
4D
17D
NEUT
(10e3/uL)
17D
Dose (ug RNA/Dose Day)
Males
Vehicle BNT16262(V9)
BNT162b3c
0
30
(b) (4
48 04
42.61
18 51
18 27
31.24
32.46
12.27
11.63
Vehicle
0
0.90x
0.90x
0 96x
1.04x
44 91
41.67
18.37
18.62
32.34
33.18
11.11
11.33
Females
BNT16262(V9) BNT162b3c
30
(b) (4)
0.93x
0.91x
0.97x
1.21x
0.27x
1.18x
392 1
178 8
7.60
3 84
1.083
0.674
301 7
168.9
0 43x
1.31x
1.41x
2.30x
6.01
2.16
1.30x
2.64x
2.28x
6.60x
0 920
0.409
2.51x
6.04×
74

BLA 125742
Dose (ug RNA/Dose Day)
Parameter
Test Article
Males
Vehicle BNT16262(V9)
0
30
BNT16263c (b) (4)
Vehicle
0
Females
BNT16262(V9) BNT16263c
30
(b) (4)
MONO
(10e3/uL)
4D
17D
0.109
0.071
1.83x
3.30x
0.093
0.056
1.89x
2.75x
EO (10e3/uL)
4D
17D
0.081
0.056
-
2.52x
0.057
0.029
-
3.17x
BASO
(10e3/uL)
4D
17D
0.016
0.003
1.88x
5.67x
0.009
0.001
1.89x
8.00x
LUC
(103/uL)
4D
17D
0.046
0.026
4.07x
8.04x
0.030
0.010
4.20x
13.20x
FIB (mg/dL)
17D
253.1
2.36x
217.2
2.49x
Control mean values and the ratio of the test article-related findings relative to control means are listed.
-= Not test article related; BASO = Basophil, absolute; D = Day; EO = Eosinophil, absolute;
FIB = Fibrinogen; HCT = Hematocrit; LUC = Large unstained cells, absolute; MCH = Mean cell hemoglobin;
MCHC = Mean cell hemoglobin concentration; MONO = Monocyte, absolute; NEUT = Neutrophil, absolute;
RDW = Red cell distribution width; RETIC = Reticulocyte, absolute; WBC = White blood cells.
Table 48: Test article-related hematology and coagulation parameter effects at main sacrifice (mean control values and ratio relative to control mean)
Dose (Ag RNA/Dose Day)
Parameter
Test Article
Males
Vehicle BNT16262(V9)
BNT16263c
30
(b) (4)
Vehicle
Females
BNT16262(V9) BNT16263c
30
(b) (4)
RDW (%)
R22
11.93
1.13x
10.80
1.21x
Control mean values and the ratio of the test article-related findings relative to control means are listed.
R = Recovery day; RDW = Red cell distribution width.
Table 49: Test article-related hematology and coagulation parameter effects at recovery phase (mean control values and ratio relative to control mean)
Bone Marrow Assessment
Bone marrow smears were prepared for all animals and were not examined.
Systemic toxicity:
No treatment-related, mortality, nor any toxicologically relevant changes in clinical signs, body weight, food consumption, body temperature, ophthalmic changes, or urinalysis were reported.
Organ Weight:
In (b) (4)
, test article-related organ weight differences included
higher absolute and relative (to body and brain weight) spleen weights were reported.
75

BLA 125742
No test article-related organ weight changes were reported at the end of the recovery phase.
Table of organ weights results for males
Group Number:
Dose:
BWT
Brain
Epididymis
Gland, Adrenal
Gland, Prostate
Heart
Kidney
Liver
Spleen
Testis
Thymus
ABS
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW-BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW:BRN
N
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
N
10
10
10
10
10
10
REF
0 ug/day
Vean Ratio
SD
296 06 R REF
16 40
1 9061 R REF 0 0899
0 6449 R REF 0 0335
1 0000 R REF 0 0000
11647 R REF 01713
0 3936 R REF 0 0536
06112 R REF 0 0867
0 0697 R REF 0 0068
0 0236 R REF 0 0021
0 0366 R REF 0 0040
0 7215 R REF 0 1036
0 2439 R REF 0 0328
03781 R REF 00476
0 9152 R REF 0 0698
0 3097 R REF 0 0260
0 4807 R REF 0 0388
2 1659 R REF 0 1836
0 7312 RREF 00411
11356 R REF 0 0682
83218 R REF 0 5205
28131 R REF 0 1435
4 3681 R REF 02325
0 5951 R REF 0 0613
0 2008 R REF 0 0147
03120 R REF 0 0264
Mean Ratio
SD
32727 RREF 03106
1 1090 RREF 01254
17171 RREE 01440
0 5914 R REF 00676
0 1999 R REF 0 0222
0 3098 R REF 0 0266
TO
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
2
30 ug/day
(b) (4)
Mean
271 17
Ratio
SD
1 9159
0 7087
1 0000
1 0626
0 92
17 12
101 0 1445
110 0 0664
100
0 0000
0 91
0 1281
(b) (4)
0 3922
1 00
0 5570
0 91
0 0727
1 04
0 0428
0 0756
0 0149
0 0267
1 13 0 0045
0 0383
1 04 0 0091
0 7324
102 0 2129
0 2699
111 0 0726
0 3808
1 01 0 0941
09242
101 0 1151
0 3405
110
0 0329
0 4852
2 2197
0 8179
1 01
0 0758
1 02
0 2229
112
0 0507
1 1600
7 7880
2 8771
4 0850
102
0 0939
0 94
0 4860
102
0 1801
004
0 3960
0 7700
0 2842
0 4019
129
0 1038
142
0 0352
129
0 0431
Mean
Ratio
SD
3 4683
106 03109
1 2803 1 15 0 1001
106
0 1262
1 8123
0 4673
0 79
0 0934
0 1718
0 86 0 0293
0 2448
0 79
0 0507
Table 50: Male's organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed as organ weight from animals taken at the end of the terminal phase.
Body weight was b) (4)
in groups (b) (4)
, respectively.
. Spleen weight was increased 29% (b) (4)
, respectively. Thymus weight was decreased 21% (b) (4) in (b) (4)
76

BLA 125742
Table of organ weights results for females
Group Number:
nse:
BWT
Brain
Gland, Adrenal
Heart
Kidney
Liver
Ovary
Spleen
Thymus
ABS
ABS
OW-BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW-BW
OW:BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW•BRN
ABS
OW:BW
OW:BRN
ABS
OW:BW
OW•BRN
ABS
OW:BW
REF
0 ug/day
N
Mean Ratio
SD
10
198 73
R REF
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
OW:BRN
10
18610 R REF 0 0694
0 9383 R REF 0 0507
1 0000 R REF 0 0000
0 0882 R REF 0 0162
0 0442 R REF 0 0068
0 0474 R REF 0 0088
0 7450 R REF 0 0803
0 3749 R REF 00343
0 4004 R REF 0 0418
15273 R REF 0 0808
0 7696 R REF 00415
08216 RREF 00519
5 4571 RREF 03313
2 7466 R REF 0 0920
2 9329 R REF 0 1468
01167 R REF 0 0158
0 0588 R REF 0 0076
0 0627 R REF 0 0079
0 4382 R REF 0 0669
0 2202 R REF 0 0294
0 2353 R REF 00333
0 4588 R REF 0 0700
0 2310 R REF 0 0336
0 2469 R REF 0 0386
N
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
TO
10
10
10
10
10
10
2
30 ug/day
Mean
Ratio
SD
10 69
194 56
0 98
1 7868
0 9203
0 98
0 0467
1 0000
100 0 0000
0 0886
100
0 0156
0 0454
103
0 0065
0 0496
105
0 0085
0 7573
102
0 0866
0 3893
104 0 0417
0 4248
1 06 0 0563
1 6343
107 0 0778
0 8412 1 09 0 0418
0 9153
111 00477
5 6490
104
0 5559
2 9002
1 06 0 1853
3 1630
108
03132
0 1053
00542
0 0590
0 6796
0 3492
0 3803
0 3967
0 2031
0 2221
0 90 0 0180
092
0 0097
0 94
0 0101
1 55
0 1031
1 59 0 0489
162
0 0550
086 0 1131
0 88
0 0583
0 90
0 0655
(b) (4)
(b) (4)
Table 51: Female's organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed as organ weight from animals taken at the end of the terminal phase.
Spleen weight was increased 55% (b) (4) in groups (b) (4)
weight was decreased 14% and (b) (4) in groups (b) (4)
, respectively. Thymus , respectively.
Gross pathology:
Dosing phase
In groups (b) (4), large draining lymph nodes (abnormal size, enlarged) and dark/pale and/or firm injection sites (abnormal color, dark/pale and/or abnormal consistency, firm) were reported.
In group (b) (4) , large spleen and inguinal lymph nodes (abnormal size, enlarged) were reported.
77

BLA 125742
Male
Female
Group Namber:
Dose: O us/dar 30 u3dar (5) (4) o uSdar 30 42/dar (b) (4)
/day
/day
10
(b) (4)
LIVER
LUNG
Abnonnal color
LYMPH NODE, DRAINING
(b) (4)
LYMPH NODE, INGUINAL
Aomonnal size
SITE, INJECTION
monomma. colo.
Abnonnal consistency
SPLEEN
Table 52: Gross findings at dosing phase
Recoverv phase
In one group (b) (4)
, large draining lymph nodes (abnormal size,
enlarged were reported. Large inguinal lymph nodes (abnormal size, enlarged were reported in one group (b) (4) , indicating a partial recovery of these findings. In groups (b) (4)
, pale/dark and/or firm injection sites and enlarged spleen were not reported at the end of recovery phase, indicating a complete recovery of these findings.
Female
Group Number:
Dose: o me day 30 weday (6) (4)
Ong/day 30 mg/day (b) (4)
Amunals Eramined
LYMPH NODE, DRAINING
Abnonnal size
LYMPH NODE, INGUINAL
Abnormal size
ADIPOSE TISSUE
Abnonnal color
Almonnel concisiones
Table 53: Macroscopic findings at recovery phase
Microscopic findings:
Terminal sacrifice
In b) (4)
, findings at the injection site (mixed cell inflammation and
edema), draining and inguinal lymph nodes (increased cellularity, plasma cells and germinal centers), liver (hepatocellular vacuolation), spleen (increased cellularity, hematopoietic cells and germinal centers), and bone marrow (increased cellularity, hematopoietic cells) were reported.
78

BLA 125742
EYE
Mineralization, Cornea
Rosettes retina
GLAND, ADRENAL
Hypertrophy, Cortex
GLAND, HARDERIAN
Degeneration/Necrosis
Infiltration mononuclear cell
GLAND, PITUITARY
Cyst
GLAND. PROSTATE
Infiltration mononuclear cell
Group Number: Male 1
2
Dose:
0 ug/day 30 ug/day
No. Animals Per Dose Group:
Number Examined
Unremarkable
Minimal
Minimal
Number Examined
Unremarkable
Present
Number Examined
Unremarkable
Minimal
Minimal
Number Examined
Unremarkable
Minimal
Number Examined
TInremarkable
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
1
10
10
Female 1
(b) (4)
0 ug/day 30 ug/day
/day
10
10
(b) (4)
10
9
1
1
10
10
10
6
2
2
3
3
10
9
1
10
10
1
10
10
9
1
10
10
3
(b) (4)
/day
10
(b) (4)-
Minimal

BLA 125742
GLAND, SALIVARY
Hypertrophy
GUT-ASSOCIATED LYMPHOID TISSUE
Mineralization, Germinal center
HEART
JOINT
Inflammation, Extra-capsular
Physeal dysplasia
KIDNEY
Tubular basophilia
Infiltration mononuclear cell
Dilatation, Pelvis
Group Number: Male 1
2
Dose:
0 ug/day 30 ug/day
No. Animals Per Dose Group:
Number Examined
Unremarkable
Minimal
Number Examined
Unremarkable
Minimal
Number Examined
Unremarkable
Number Examined
Unremarkable
Minimal
Minimal
Number Examined
Unremarkable
Minimal
Minimal
Minimal
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
7
3
3
10
Female 1
(b) (4)
0 ug/day 30 ug/day
/day
10
10
'(b) (4)
10
9
1
1
8
8
55
10
9
1
1
10
8
2
2
10
10
10
10
1
10
10
10
2
2
10
6
3
3
3
b) (4)
/day
10
(b) (4}
80

BLA 125742
LARGE INTESTINE, COLON
Infiltration mixed cell, Mucosa
LIVER
Vacuolation, Hepatocyte; Periportal
LUNG
Infiltration mixed cell
LYMPH NODE, DRAINING
Increased cellularity, Plasma cell
Increased cellularity, Germinal center
LYMPH NODE. INGUINAL
Increased cellularity, Germinal center
Increased cellularity, Plasma cell
Group Number: Male 1
2
Dose:
0 ug/day 30 ug/day
No. Animals Per Dose Group:
Number Examined
Unremarkable
Minimal
Number Examined
Unremarkable
Minimal
Number Examined
Unremarkable
Minimal
Number Examined
Unremarkable
Minimal
Mild
Moderate
Minimal
Mild
Number Examined
Unremarkable
Minimal
Mild
Minimal
10
10
10
10
10
10
10
10
8
10
10
10
10
5
5
5
10
10
4
2
6
2
4
10
5
5
Female 1
(b) (4)
0 ug/day 30 ug/day
/day
10
10
(b) (4)
10
10
10
10
10
9
1
1
10
8
10
9
1
10
10
10
10
10
10
10
9
10
1
9
1
7
5
3
10
4
6
3
3
2
2
3
(b) (4)
/day
10
• (b) (4) -
81

BLA 125742
PANCREAS
Atrophy, Acinar cell
Infiltration mononuclear cell, Interstitium
SITE. INJECTION
Inflammation
Edema
SPLEEN
Increased cellularity, Germinal center
Increased cellularity, Hematopoietic cell
STOMACH
Infiltration mononuclear cell, Serosa
Erosion
Table 54: Microscopic findings at terminal sacrifice
Group Number: Male 1
2
Dose:
0 ug/day 30 ug/day
No. Animals Per Dose Group:
Number Examined
Unremarkable
Minimal
Minimal
Number Examined
Unremarkable
Minimal
Mild
Moderate
Mild
Moderate
Number Examined
Unremarkable
Minimal
Minimal
Number Examined
Unremarkable
Minimal
Minimal
10
10
10
10
10
10
10
10
10
10
10
0
10
7
3
9
1
10
5
5
10
10
10
10
Female 1
(b) (4)
0 ug/day 30 ug/day
/day
10
10
(b) (4)
10
10
10
5
5
§
10
10
10
10
10
10
6
4
1
10
10
7
3
10
g
1
10
6
6
9
g
10
9
3
(b) (4)
/day
10
76) (4)-
82

BLA 125742
Recovery sacrifice
A complete recovery of most of the findings reported at the terminal phase. Inflammation at the injection site was characterized by mostly lymphocytes and plasma cells with few neutrophils (indicating partial recovery) and no edema (full recovery). In b) (4) increased cellularity of the germinal centers in the spleen partially recovered, as the incidence and or severity of these findings were lower in recovery phase animals as compared with dosing phase animals. At the end of recovery phase, mature plasma cells had replaced the plasma blasts identified in the inguinal and draining lymph nodes in the dosing phase animals. Infiltration of macrophages was reported in the draining lymph nodes (minimal to mild) in b) (4)
and in the inguinal lymph nodes (minimal) of group 2 males and females.
Dermal Assessment
Dosing phase
In all group 2 (except animal #17) animals, related injection site edema grade 2 (slight, edges of area well defined by definite raising) or grade 3 (moderate, raised approximately 1 mm) were reported following dosing on days 1, 8 and or 15. The edema was generally reported up to 72 hours post dose, and fully resolved prior to dose administration on days 8 and 15. In all group 2 (except animals 16-21 and 30) animals, erythema was also reported at the injection site, following each dose administration. However, it was only a grade 1 (very slight, barely perceptible and fully resolved prior to the next dose administration.
In all group 3 (b) (4)

BLA 125742
Group mean dermal assessment data are listed in the table below:
Male
Parameter
Edema - Left
Parameter
Erythema - Left
Phase
Dosing
Dosing
Dosing
Phase
Dosing
Dosing
Dosing
Dav
1
Group
1: Saline
2: BNT16262 (V9)
3: BNT16263c
8
1: Saline
2: BNT16262 (V9)
3: BNT16263c
15 1: Saline
2: BNT16262 (V9)
3: BNT16263c
Male
Day Group
1: Saline
2: BNT16262 (V9)
3: BNT16263c
8
1: Saline
2: BNT16262 (V9)
3: BNT16263c
15
1: Saline
2: BNT16262 (V9)
3: BNT16263c
N
15
15
15
15
15
15
15
15
15
N
15
15
15
15
15
15
15
15
15
Standard Pairwise
Mean Deviation p-value
0.00
0.00
REF
0.63
0.51
0.001 **
(b) (4)
0.00
1.19
(b) (4)
0.00
0.51
REF
0.001 **
0.00
1.33
b) (4)
0.00
REF
0.45
0.001 **
Standard Pairwise
Mean Deviation p-value
0.00
0.00
REF
0.03
0.13
0.682
(b) (4)
0 00
0.00
0.27
REF
0.001 **
0.23
[6) (4)
000
0.00
(b) (4)
0 00
0.00
REF
0.999
Table 55: Edema and erythema findings in males at study days 1, 8, and 15
84

BLA 125742
Female
Parameter
Edema - Left
Phase
Dosing
Dosing
Dosing
Dav
1
Group
1: Saline
2: BNT16262 (V9)
3: BNT16263c
1: Saline
2: BNT16262 (V9)
3: BNT16263c
15 1: Saline
2: BNT16262 (V9)
3: BNT16263c
Female
N
15
15
15
15
15
15
15
15
15
Standard Pairwise
Mean Deviation p-value
0.00
0.00
REF
1.28
0.57
0.001 **
(b) (4)
0.00
1.44
(b) (4)
000
1.64
(b) (4)
0.00
0.23
0.00
0.34
REF
0.001 **
REF
0.001 **
Parameter
Erythema - Left
Phase
Dosing
Dosing
Dosing
Day Group
1: Saline
2: BNT16262 (V9)
3: BNT16263c
8
15
1: Saline
2: BNT16262 (V9)
3: BNT16263c
1: Saline
2: BNT16262 (V9)
3: BNT16263c
N
15
15
15
15
15
15
15
15
15
Table 56: Edema and erythema findings in females at study days 1, 8, and 15
Standard Pairwise
Mean Deviation p-value
0.00
0.00
REF
0.56
0.38
0.001 **
(b) (4)
000
0 50
(b) (4)
0 00
0.33
(b) (4)
0.00
0.09
0.00
0.22
REF
0.001 **
REF
0.001 **
85

BLA 125742
Male
Parameter
Phase
Edema - Left
Recovery
Group
2: BNT16262 (V9)
3: BNT16263c
N
4
5
Standard
Mean Deviation
1.08
0.17
(b) (4)
Erythema - Left
Recovery
2: BNT16262 (V9)
3: BNT16263c
4
5
0.00
(b) (4)
0.00
Female
Parameter
Phase
Edema - Left
Recovery
Group
2: BNT16262 (V9)
3: BNT16263c
N
5
5
Standard
Mean Deviation
1.07
0.15
(b) (4)
Erythema - Left
Recovery
2: BNT162b2 (V9)
3: BNT16263c
5
5
0.13
(b) (4)
0.18
Table 57: Edema and erythema findings in males and females at recovery phase
Body temperature:
No test article-related effects on body temperature was reported.
Urinalysis:
There were no test article-related findings on urinalysis. Due to small magnitude of the difference and general overlap in magnitude of individual values with controls, all statistically significant or apparent differences in urinalysis parameters between groups 2 and 3 and control group were not test article related.
Male
Gronn Number
Phase
Day
Dose:
REF
O jag/day
2
30 ug/day
3 (b)
(4)
pH (one)
Dosing
Recovery
22
SG
Dosing
17
Recovery
22
VOLUME (mL)
Dosing
Recovery
22
Mean (10) 7.10
SD
039
Mean (5)
730
SD
0.45
Mean (10) 1.0322
SD
0.0205
Mean (5) 1.0556
SD
0.0038
Mean (10)
14.90
SD
15 54
Mean (5)
3.70
SD
007
(10)
6.75
0.35
7.20
0.27
1.0260
0.0227
(5
1.0340
0.0146
(10)
17.80
16.95
(5
8.20
5.50
(10)
(5)
(10
(5)
(10)
(5
Table 58: Urinalysis for male groups
86

BLA 125742
Female
Group Number:
Phase
Day
Dose:
REF
0 ug/day
2
30 ug/day
з
(b) (4)
pH (one)
Dosing
Recovery
22
SG (one)
Dosing
Recovery
22
VOLUME (mL)
Dosing
Mean (10) 6.75
SD
0.26
Mean (5)
7.00
SD
0.61
Mean (10) 1.0243
SD
0.0128
Mean (5) 1.0240
SD
0.0174
Mean (10)
9 00
SD
7.03
(10)
(5
6.20 T
0.26
6.60
0.65
(10)
(5)
(10)
1.0288
0.0164
15 1.0364
0.0177
(10)
9.60
9.05
(10)
(5)
(10)
Recoserv
Mean (5)
SD
11.00
1.38
(5
6.00
5.09
(5)
Table 59: Urinalysis for male groups
Serology:
Microneutralization (MN assay for serological detection of SARS-CoV-2 specific neutralizing antibodies in animal sera were used. This is relative to the "work order 4" agreed between (b) (4) and Pfizer. The b) (4) method is a specific technique used for the (b) (4)
The following table shows
geometric mean titers for grouped subiects by sex and for vaccine administered.
Study Day
Sex
saline
PIO Day 8
(Day -5)
Day 17
Male
Female
Male
Female
R:P Day 21
Male
(Day 38)
Female
5
5
5
5
5
5
PIO = prior to dose initiation; RP = Recovery phase
30ug
(b) (4)
BNT16262(V9) BNT16263c
5
(b) (4)
§
1114
2501
5120
5120
Table 60: Geometric mean titers (GMTs) for each dose group by sampling day and sex
In (b) (4)
SARS-CoV-2 neutralizing antibody responses in males and females at the end
of the dosing (day 17) and recovery phases (day 21) were reported. SARS-CoV-2 neutralizing antibody responses were not reported in animals prior to vaccine administration or in group 1 (control) animals.
Test article related effects are listed in the table below:
Test article related effects
1 Albumin ^ Globulin
I I AG ratio
87

BLA 125742
Test article related effects
1 Reticulocytes ^ Monocytes
^ Neutrophils
^ Eosinophils
^ Basophils
^ WBC
^ LUC
^ Fibrinogens
^ Red cell distribution width (RDW%)
^ Alphal-acid glycoproteins ^ Alpha2-macroglobulins
^ Spleen weight
¿ Thymus weight for females
Injection site findings (mixed cell inflammation and edema)
Draining and inguinal lymph nodes findings (increased cellularity, plasma cells and germinal centers)
Liver findings (hepatocellular vacuolation)
Spleen findings (increased cellularity, hematopoietic cells and germinal centers)
Bone marrow (increased cellularity, hematopoietic cells)
Immune responses in b) (4)
Assessment:
No treatment-related, mortality, nor any toxicologically relevant changes in clinical signs, body weight, food consumption, body temperature, ophthalmic changes, or urinalysis were reported.
Minimal decreases in globulin concentration was reported in b) (4)
Concurrently, minimally increased albumin was reported. Hence, the albumin to globulin ratio was lower in (b) (4)
from (b) (4)
. These changes indicate an acute phase
response/inflammation. These changes were not reported in the recovery animals.
Reticulocytes are immature red blood cells (RBCs. In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow and then circulate for about a day in the blood stream before developing into mature red blood cells. Like mature red blood cells, in mammals, reticulocytes do not have a cell nucleus.26 Abnormally low numbers of reticulocytes can be attributed to chemotherapy, aplastic anemia, pernicious anemia, bone marrow malignancies, problems of erythropoietin production, various vitamin or mineral deficiencies (iron, vitamin B12, folic acid), disease states (anemia of chronic disease) and other causes of anemia due to poor RBC production. 27
Monocytosis could be indicative of the intended immune response or could be secondary to muscle damage at the site of iniection as an indication of inflammation and repair. The increases in the monocyte count might be related to test article treatment.
26 https://en.wikipedia.org/wiki/Reticulocyte
27 https://www.uofmhealth.org/health-library/hw203366
88

BLA 125742
Neutrophils are key components in the system of defense against infection. An individual with absence or scarcity of neutrophils (neutropenia) is vulnerable to infection. The increase in neutrophils might be related to the immune responses initiated by the test article treatment.
Eosinophils are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood.
Basophils play a role in both parasitic infections and allergies. Basopenia has been reported in association with autoimmune urticaria.
White blood cells (WBCs) (also called leukocytes or leucocytes) are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic system.28 The increase in WBC might be related to the immune response induced by the test article treatment.
LUC is a measurement of the large, peroxidase-negative cells which cannot be further characterized (i.e. as large lymphocytes, virocytes, or stem cells) present in a biological specimen. In LUC are found large lymphoid cells, more immature lymphocytes and other cells.
If the value is higher than normal, blood counts should be checked under a microscope slide.
The increases in fibrinogen levels were not considered frank toxicity but rather an anticipated effect associated with an immunological response.
A red cell distribution width (RDW) test is a measurement of the range in the volume and size of red blood cells (erythrocytes). Red blood cells move oxygen from lungs to every cell in the body.
The RDW blood test is often part of a complete blood count (CBC), a test that measures many different components of the blood, including red cells. The RDW test is commonly used to diagnose anemia, a condition in which the red blood cells can't carry enough oxygen to the rest of the body. The RDW test may also be used to diagnose?9.
1- Other blood disorders such as thalassemia, an inherited disease that can cause severe anemia
2- Medical conditions such as heart disease, diabetes, liver disease, and cancer, especially colorectal cancer.
Alpha-1-acid glycoprotein (alAGp,30 AGP or AAG), which is modulated by two polymorphic genes, is an acute phase (acute phase protein) plasma alpha-globulin glycoprotein. It has a
28 Maton, D., Hopkins, J., McLaughlin, Ch. W., Johnson, S., Warner, M. O., LaHart, D., & Wright, J. D., Deep V.
Kulkarni (1997). Human Biology and Health. Englewood Cliffs, New Jersey, US: Prentice Hall. ISBN 0-13-981176-1.
29 https://medlineplus.gov/lab-tests/rdw-red-cell-distribution-width/ 30 https://en.wikipedia.org/wiki/Orosomucoid#cite_note-loganabbrev-1
89

BLA 125742
normal plasma concentration between 0.6-1.2 mg/mL (1-3% plasma protein) and is synthesized primarily in hepatocytes (5). Plasma levels are affected by pregnancy, burns, certain drugs, and certain diseases, particularly HIV (5). The function of alpha-1-acid glycoprotein is to act as a carrier of basic and neutrally charged lipophilic compounds. It is known as the primary carrier of basic (positively charged) drugs (whereas albumin carries acidic (negatively charged) and neutral drugs), steroids, and protease inhibitors (5, 6). AGP shows a complex interaction with thyroid homeostasis. Alpha-1-acid glycoprotein (in low concentrations) was reported to stimulate the thyrotropin (TSH) receptor and intracellular accumulation of cyclic AMP. However, high AGP concentrations inhibited TSH signaling (7, 8). Alpha-1-acid glycoprotein has been identified as one of four potentially useful circulating biomarkers for estimating the five-year risk of all-cause mortality (the other three are albumin, very low-density lipoprotein particle size, and citrate) (9).
Alpha-1-acid glycoprotein increases in obstructive jaundices while diminishes in hepatocellular jaundice and in intestinal infections. 31
Alpha-2-macroglobulin (a2M) is a large plasma protein found in the blood, mainly produced by the liver, and also locally synthesized by macrophages, fibroblasts, and adrenocortical cells. It acts as an antiprotease and is able to inactivate an enormous variety of proteinases. It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein and as an inhibitor of coagulation by inhibiting thrombin. Because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, TGF-B, insulin, and IL-1B, it may act as a carrier protein. In the nephrotic svndrome when other lower molecular weight proteins are lost in the urine, the concentration of alpha-2-macroglobulin rises 10-fold or more32
In (b) 4)
all clinical pathology findings (type and magnitude) were generally similar,
and consistent with expected immune responses to vaccines or secondary to inflammation. In both sexes, the main findings were present on days 4 and/or 17 and included higher acute phase proteins (alpha-1 acid glycoprotein; 7.0x-42x controls], alpha-2-macroglobulin (3.3x-128x] and fibrinogen [2.4x-2.6x]) and white blood cell count (1.28x-2.95x; primarily involving neutrophils, monocytes and large unstained cells, which typically represent large mononuclear cells) and lower albumin: globulin (0.90×-0.82x). On peripheral blood smears, hyper-segmented neutrophils present and were considered to be secondary to the robust increases in neutrophil counts and likely related to mobilization of bone marrow storage neutrophils and prolonged neutrophil lifespan in circulation (10). These findings were consistent with the immune responses to vaccines.
Spleen weight increase might be related to the intended immune response. The spleen plays important roles in regard to red blood cells and the immune system?3
. It removes old red blood
cells and holds a reserve of blood in case of hemorrhagic shock while also recycling iron. As a part of the mononuclear phagocyte system, it metabolizes hemoglobin removed from senescent erythrocytes. The globin portion of hemoglobin is degraded to its constitutive amino acids, and the heme portion is metabolized to bilirubin, which is subsequently shuttled to the liver for
31 https://en.wikipedia.org/wiki/Orosomucoid
32 https://en.wikipedia.org/wiki/Alpha-2-Macroglobulin
33 Spleen, Internet Encyclopedia of Science.
90

BLA 125742
removal34. It synthesizes antibodies in its white pulp and removes antibody-coated bacteria along with antibody-coated blood cells by way of blood and lymph node circulation.
The thymus is a specialized primary lymphoid organ of the immune system. Within the thymus, T cells or T lymphocytes mature. T cells are critical to the adaptive immune system, where the body adapts specifically to foreign invaders. The thymus is composed of two identical lobes and is located anatomically in the anterior superior mediastinum, in front of the heart and behind the sternum. 35 One of the major characteristics of vertebrate immunology is thymic involution, the shrinking of the thymus with age, resulting in changes in the architecture of the thymus and a decrease in tissue mass.36 T-cells are named for the thymus where T-lymphocytes migrate from the bone marrow to mature. Its regression has been linked to the reduction in immunosurveillance in the elderly. 37
Test article-related injection site findings (mixed cell inflammation and edema) were reported.
Inflammation is a relatively common occurrence as part of the acute phase response following administration of some vaccines.
The microscopic findings included minimally increased cellularity of hematopoietic cells (primarily myeloid) in the bone marrow and the spleen, minimal to moderate mixed cell inflammation at the injection site and increased cellularity in germinal centers of lymphoid organs. In addition, lower reticulocyte counts on day 4 (0.44x-0.27x), and higher reticulocytes on day 17 (1.20x-1.31x; females only), with minor lower red cell mass on days 4 and 17 (HCT;
0.93x-0.89x) were reported. Lower reticulocytes levels were interpreted to be a transient effect of innate immune responses (11-14).
At the terminal phase, test article-related findings in the lymph nodes (increased cellularity of plasma cells [minimal to moderate] and germinal centers [minimal to mild]), spleen (increased cellularity of hematopoietic cells [minimal] and germinal centers [minimal]), and the bone marrow (minimal increased cellularity of hematopoietic cells) were reported. This is considered secondary to immune activation and/or inflammation at the injection site. The presence of plasma cells (interpreted as plasma blasts) in the draining and inguinal lymph nodes was interpreted to reflect a robust immunological response to the vaccines. These findings correlated with macroscopic findings of abnormal size (enlarged) in the lymph nodes and spleen and increased spleen weights.
Minimal portal hepatocyte vacuolation finding was not associated with hepatic tissue damage or liver enzyme alterations. This change may be related to hepatic clearance of the pegylated lipid in the LNP (15). This finding was completely recovered at the end of 3-week recovery phase.
Test article-related immune responses in groups 2 and 3 were reported.
34 Mebius RE, Kraal G. (2005). Structure and function of the spleen. Nat Rev Immunol. 5(8):606-16.
35 https://en.wikipedia.org/wiki/Thymus.
36 Shanley D.P.; Danielle A. W.; Manley N.R.; Palmer D.B.; et al. (2009). "An evolutionary perspective on the mechanisms of immunosenescence". Trends Immunol. 30 (7): 374-381. doi: 10.1016/j.it.2009.05.001
PMID 19541538
37 Linton P.J.: Dorshkind K. (2004). "Age-related changes in lymphocyte development and function". Nat. Immunol.
5 (2): 133-139. doi: 10.1038/ni1033. PMID 14749784
91

BLA 125742
Based on the overall findings in this study, it can be concluded that in Wistar rats, repeat dose on study days 1, 8, and 15 had no adverse effects in terms of systemic toxicity at the dose level of 30 g/animal. However, due to the significant decrease in the reticulocyte levels, hematology results should be closely monitored during any clinical trial.
GLP study deviations or amendments: No significant deviations have occurred during the study that could have impacted the generated results.
Investigators Brochure: Having read and evaluated the Investigators Brochure, is it a fair, objective and reasonable summary of the toxicology data - yes ) or no (X).
Internal Communication:
Due to the significant decreases in the reticulocyte's levels, close monitoring to the hematology data in any clinical trial is highly recommended.
Communication to sponsor:
Please add the finding of this study to your Investigators Brochure.
Conclusions:
Based on nonclinical toxicity assessments, there are no significant safety issues to preclude the IND from going into effect.
92

BLA 125742
Study number 3 (Reproductive Toxicology Study):
Title and study number: A Combined Fertility and Developmental Study Including Teratogenicity and Postnatal Investigations) of BNT1621, BNT162b2 and BNT162b3 by the Intramuscular Administration in the Wistar Rat. Study number: 20256434
Performing laboratory: (b) (4)
Study initiation date: July 27, 2020
Final Report date: December 15, 2020
Test article batch/lot:
Test Item 1
Identification:
BNT162b1
Alternate Identification:
Batch No.:
(b) (4)
Lot No.:
Physical Description:
Test item identification
Test Item 2
BNT16262
CoVVAC (b) (4)
CoVAC/270320
White to off-white suspension
27 Nov 2020
Test Item 3
BNT16263
(b) (4)
Expiry Date:
Correction Factor:
Concentration
(RNA Content):
Storage Conditions:
Provided by:
None
(b) (4)
Temperature set to maintain -80°C
Sponsor
Table 61: Test item identification
Identification:
Alternate
Identification:
Batch/Lot Nos.:
Expiry Dates:
Storage Conditions:
Provided by:
N/A: Not Applicable.
Table 62: Control item identification
Animal species and strain: (b) (4) :WI(Han) Wistar rat
Breeder/supplier: b) (4)
Number of animals per group and sex:
Caesarean subgroup: 88 virgin mated females.
Control item identification
Control Item
Sterile phvsiological saline (0.9% NaCI)
NIA
(b) (4)
30 Apr 2022 and 30 Nov 2022 respectively
Ambient temperature
Test Facility
93

BLA 125742
Littering subgroup: 88 virgin mated females.
Age:
Females: 11 weeks old.
Males: 11 weeks old.
Body weight range:
Females: 179.3 - 265.4 g.
Males: 328.4 - 415.9 g.
Route and site of administration: Intramuscular injection into the quadriceps alternating on each dosing occasion.
Volume of iniection: The dose volume was 0.06 mL per injection
Frequency of administration and study duration:
Pre-mating period: Study days 1 (21 days before mating, M-21) and 8 (14 days before mating, M-14) and on gestation days (GD's 9 and 20.
Dose: 0.5 mg/mL
Stability: Analysis of stability, homogeneity and concentration of the test article under test conditions was not performed as part of the study. Stability studies were performed by the sponsor of the IND. Stability data will be reported in the final study report. The following stability data were reported:
1- Stable at a concentration of 0.5 mg/mL for 12 weeks at -80°C.
2- Stable at a concentration of 0.5 mg/mL for at least 1 month at room temperature (information provided by the study sponsor on 03 Dec 2020)
3- Homogenous for at least 6 hours following gentle inversion.
Means of administration: Intramuscular
Report status: Final report
Experimental design:
Animals were randomized and assigned to 4 different groups. Each group consisted of 22 females. Animals were administered 4 doses of saline or test article, study day 1 (21 days before mating, M-21) and day 8 (14 days before mating, M-14) and on gestation days 9 and 20.
Animals will be euthanized according to the following schedule:
FO Females: Caesarean subset: On GD21.
Littering subset: After weaning of the F1 pups (females that fail to produce a viable litter by GD26 will be euthanized and necropsied).
Unmated Females: After completion of the mating period.
Pups: On PND4 (unselected pups) or on PND21.
The details of the study design are listed in the following table:
Experimental design of the FO generation
Group
No.
Test
Material
Dose (ug mRNA)
Dose
Volume (mL)
Dose
Concentration (mg/mL)
Control item
0
0 06
0
Number and Identification of Animals
Caesarean
Littering
Subgroup
Subgroup
22
22
(1 to 22)
(201 to 222)
94

BLA 125742
Group
No.
Test
Material
Dose (ug mRNA)
Dose
Volume (mL)
Dose
Concentration (mg/mL)
2 BNT16261 (b) (4)
3
BNT16262
30
0.06
0.5
22
(45 to 66)
4 BNT16263 (b) (4)
(b) (4)
Identification of untreated males: 301 to 388.
Table 63: Experimental design of the F0 generation
Methods:
Randomization procedure: Yes.
Statistical analysis plan: Yes.
The following parameters will be evaluated:
In-life procedures, observations, and measurements
General in-life assessments - untreated males and F0 females
Frequency
Parameter
Population(s)
(Minimum required)
Mortality
All animals
At least twice daily' (at beginning and end of working day)
F1 pups will be counted daily during the preweaning phase
Cageside
Observations
All animals
All animals
Detailed Clinical
Observations
Individual
Body Weights
AlI FO females
Before and at least once on dosing davs
For males, at least 1 observation will be recorded before mating
At least once daily on non-dosing days
A full clinical examination will be performed weekly during the pre-mating period then on each weighing day during the gestation and lactation periods
Each FO female will be weighed at least weekly during pretest, twice weekly before mating and for the periods:
GDO, GD6, GD9, GD12, GD15, GD18 and GD21
LD1, LD4, LD7, LD10, LD14, LD17
and LD21
During the lactation phase, offspring were weighed on PND1, PND4, PND7, PND10, PND14, PND17 and PND21.
Number and Identification of Animals
Caesarean
Subgroup
Littering
Subgroup
22
(245 to 266)
Comments
Animals will be observed within their cage unless necessary for identification or confirmation of possible findings
Animals will be observed within their cage unless necessary for identification or confirmation of possible findings
Animals will be removed
from the cage
Animals may be weighed more often if necessary, in order to monitor health status
95

BLA 125742
Parameter
Population(s)
All F0 males
Frequency
(Minimum required)
Each FO male will be weighed at least weekly
Comments
Food Consumption
All FO females
Food consumption of each animal will be recorded at least once weekly from
Day 1 and for the periods:
GDO to GD6, GD6 to GD9, GD9 to GD12, GD12 to GD15, GD15 to GD18 and GD18 to GD21 LD1 to LD4, LD4 to LD7, LD7 to LD10, LD10 to LD14,
LD14 to LD17 and LD17 to LD21
Quantitatively measured
Estrous cvcles will be monitored
Estrous Cycles
All FO Females pre-dosing (2 weeks), then for
2 weeks before mating and during cohabitation until confirmation of
GDO
Animals will be paired on the basis
Mating
Males and all FO
Females
of 1 male and 1 female for a maximum of 14 davs
The day of mating will be confirmed by the presence of sperm in a vaginal smear or a vaginal plug and will be recorded and taken as day 0 of gestation (GDO)
The same untreated males will be used to mate both subgroups
a. Except on davs of receipt and necropsy where frequency will be at least once daily.
Table 64: General in-life assessments - untreated males and F0 females
Animals are removed from
the cage
Mated females will be separated from the male once mating has been confirmed and smearing will cease or when the appearance of the female suggests pregnancy from an undetected mating
Pregnancy and parturition (littering subset only)
For each FO female, the following will be recorded:
1- Date of mating (GDO).
2- Date of parturition (LDO).
3- Duration of gestation.
4- Abnormalities of nesting or nursing behavior.
5- Number of implantation sites at necropsy after staining with ammonium sulphide solution).
Litter data (littering subset only)
Population(s)
Each Litter
Litter data
Frequency/Comments
Number of pups born (live and dead)
External abnormalities of the pups
Number and sex of pups alive on PND1, PND4, PND7, PND10, PND14, PND17 and PND21
Physical development of the offspring, as assessed by the intra-litter onset and duration of pinna unfolding from PND1 and eye opening from PND12
Pupillary reflex and auditory reflex on PND21
External and necropsy findings of dead pups
96

BLA 125742
The size of each litter will be adjusted to 8 pups on PND4 by eliminating extra pups by random selection to yield where possible 4 male and 4 female pups per litter. Extra pups will be euthanized by an intraperitoneal injection of sodium pentobarbitone.
Antibody evaluation
Antibody sample collection
Bioanalytical sample collection
Group
Nos.
1 to 4
Number of Females
Pre-dose on Days of Dosing
Pretest
MOa
Necropsy (GD21 or LD21/PND21)b
All FO females
X
X
Selected fetuses from all litters
1 to 4
of caesarean subset
Selected pups (1 male and
1 to 4
1 female if possible) from all litters of the lactation subset
Unscheduled euthanasia
Х
(when possible, done in the animal facility
X: Sample collected; -: Not collected.
MO: First day of pairing; GD: Gestation day; LD: Lactation day; PND: Post natal day.
a: Sample collected just before pairing.
b: The day of necropsy (i.e., (b) (4)
); on GD26 for not pregnant F254 (BNT162b2, 30 g); (b) (4)
X
X
X
Method/Comments:
Target Volume (mL):
Anticoagulant:
Special Requirements:
Processing
FO females: Jugular vein
Fetuses: Small incision after anesthesia
Pups: Intracardiac
Target 0.5 mL for FO females
Target 0.3 mL pooled per litter for fetuses
Targeted 0.5 mL pooled per litter from 2 pups (ideally 1 male and 1 female)
None
None
Serum
Unscheduled necropsy
Animals
Not Mated Females
Mated Females
Examination
Full macroscopic examination of the thoracic and abdominal cavities, including the injection sites. Any abnormalities observed will be sampled and preserved Full macroscopic examination of the thoracic and abdominal cavities, including the injection sites, to determine their pregnancy status, number of corpora lutea and numbers and types of uterine implantations Any abnormalities observed will be sampled and preserved. Any fetuses from these females will not be examined and discarded
Scheduled euthanasia
Surviving animals will euthanized by carbon dioxide inhalation and exsanguination (with the exception of the PND4 extra pups) and then necropsied according to the following schedule:
FO females: Caesarean subgroup: On GD21.
F43 that failed to mate was euthanized after the mating period (on day 43).
97

BLA 125742
Littering subgroup: On LD21, after weaning of the F1 pups.
F226 and F254 that failed to produce a viable litter by GD26 or GD27 were euthanized and necropsied; F277 with a mistimed pregnancy (mating not detected) was euthanized and necropsied after the end of the mating period on day 43).
Culled F1 pups: On PND4.
Euthanized F1 pups: On PND21.
Necropsy
Caesarean subset
All animals will be submitted to a full macroscopic examination of the abdominal and thoracic cavities including the injection sites. Any abnormalities observed will be recorded and preserved but not examined further in first instance. For each female euthanized on GD21, the ovaries and uterus will be removed and examined including examination of the placentae. The following data will be recorded:
Necropsy data
Parameters
Comments
Pregnancy status
Gravid uterus weight
Number and distribution of intrauterine implantations
Number of corpora lutea
The uterus of apparently non-pregnant females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites
Classified as: Live fetuses, dead fetuses, early resorptions and late resorptions
Fetal weights
Individual weights were recorded
Fetal sex
-: No comment.
Subgroup 2 (Natural delivery)
The carcasses of PND21 pups were preserved for possible skeletal examinations. No further examination was performed.
For all FO females, the number of implantation sites were recorded.
Fetal examination
Each fetus was examined for external defects and euthanized by oral administration of sodium pentobarbitone. Approximately one half of each litter was submitted to fresh visceral examination of the body (abdominal and thoracic cavities). The head was fixed in Harrison's fluid for subsequent examination by serial sectioning. The remaining carcass was retained and fixed in ethanol.
The remaining half of the fetuses in each litter was eviscerated and then processed for skeletal examination. The skeletal examinations were performed following maceration of the soft tissues with aqueous potassium hydroxide, staining of the skeleton with Alizarin red then passage into glycerol. Soft tissue and skeletal examinations were performed using a binocular microscope.
98

BLA 125742
Results:
Serum Antibody Analysis
In groups, 3, () (4), administration of 4 doses (2 prior to mating and 2 during gestation) of the test article elicited SARS-CoV-2 neutralizing antibody responses in the majority of females just prior to mating (M-14), at the end of gestation (GD21), and at the end of lactation (LD21). In most offspring (fetuses on GD21 and pups on PND21), SARS-CoV-2 neutralizing titers were also detected. In animals prior to vaccine administration or in saline-administered control animals, SARS-CoV-2 neutralizing antibody titers were not reported.
The following table shows geometric mean titers (GMT) by time-point
(Interval/Occasion) and by group of females or offspring (fetuses and pups).
Interval/Occasion
Pretest
MO
GD21 (Dams)
LD21
Fetuses (GD21)
Pups (PND21)
Time-point legend:
MO= just prior to mating
GD21 = gestation day 21
LD21= lactation day 21
PND21= post-natal days
Saline
BNT162b1
BNT16262
BNT162b3
5.0
[(b) (4)
5.3
(b) (4)
5.0
3886.4
5.0
3445.5
5.0
3620.4
5.0
640.0
5.0
4561.4
These GMTs exclude values from no pregnant females and other intermittent sample time points .
See Appendix 1 footnotes for list of all excluded samples, in data table they are marked (*)
Table 65: Geometric mean titer by time-point and by group of females or offspring (fetuses and pups)
Mortality
No test article-related death was reported. (b) (4)
. One group 3 animal delivered 8 stillborn pups. All
these findings were not different than that reported in the historical data. Such cases of total litter death at or shortly after birth are present in the historical control data (2 studies (A19 in 2019 and V17 in 2017) out of 18 between 2015 and 2019).
Clinical observations
No adverse clinical signs during the premating and gestations periods related to any of the 3 vaccine candidates were reported.
Swelling (associated or not with limping and/or piloerection for 1 or 2 days after the second dose onlv) was reported at the injection site of groups
3, (b) (4) animals on mating day 21 (M-21),
M-14, gestation day 9 (GD9) and GD 20.
No adverse clinical signs during the lactation period related to any of the 3 vaccine candidates were reported.
99

BLA 125742
Abnormal vocalization, chromodacryorrhea, desquamation, erythema, localized hair loss, malocclusion, long or missing teeth, red vaginal discharge, red stained fur, scabs), sore(s) were reported sporadically across the groups. These findings were considered to be incidental, related to the method of dose administration or to the pregnancy status of the females.
Body weight and food consumption
No test article-related body weight changes or food consumption was reported.
In groups (b) (4)
, compared with the control
group (33 g) throughout the lactation phase. This was not considered vaccine-related, but due to an atypical high value in the control group compared with the historical control data range (from (b) (4)
).
Estrous Cycle Data
No test article-related effect on the estrous cycle was reported.
Parameter
Cycle
Irregularity index
Percentage of estrus days
Percentage of females acyclic or with acyclic period
(days)
Group 1, Control, 0 ug
MEAN
SD
4.02
0.19
0.30
44
26.95
Group 2, BNT16261. (b) (4)
MEAN
SD
(b)
（4
Group 3, BNT16262, 30 ug
MEAN
SD
4.00
42
0.18
0.30
42
26.70
5.00
Group 4. BNT 18263. (6) (4)
MEAN
SD
N
b) (4)
Table 66: Mean estrous cycle data - Before dosing
Parameter
Cycle length (days)
Irregularity index
Percentage of estrus days
Percentage of females acyclic or with acyclic period
Group 1, Control, 0 ug
MEAN
SD
N
Group 2 BNT18261. (b) (4)
MEAN
SD
4.00
0.00
38
38
zE
88=
18.2
(b) (4)
Group 3, BNT16262, 30 pg
MEAN
SD
4.02
01
38
0.05
0.12
38
3.68
18.2
GrOUp 4 BNT16263, (b) (4)
MEAN
SD
b) (4)
Table 67: Mean estrous cycle data - Pre-mating period
Maternal Mating Performance and Fertility
100

BLA 125742
No test article-related effects on mating performance or fertility was reported.
(b) (4)
In total (caesarean and littering subgroups combined), 44,
1,44 and wid
(out of 44) females
mated in groups 1,
", 3, and &
, respectively (including F277, from group, not detected at the
time of mating).
Therefore, the copulation index was 100, (6) (4), 100, and (b) (4) in groups 1, %
', 3, and &
respectively.
Mated females (majority) were inseminated within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was consequently 3.0, (b) (4)
, 2.8
and (* (4)
days in groups 1, 8, 3, and, respectively. In total, there were 43, 41, 42, and 44 pregnant females autos. 43 per group nares is croups 5 l.2.3.ah, respectivels. Therefore the In total, there were 43/44, (6) (4), 42441 and (6) (4) pregnand/mated females in groups 1,%, 3, and
', respectively. Therefore, the fertility index was 98%, 95%, (b) (4) and (b) (4) in groups 1, ® and®, respectively.
GROUP
DOSING
1
2
3
Control BNT16201 BNT162b2 BNT1623
O ug (b) (4)
30 ug (b) (4)
LITTERING AND CAESAREAN SUBSETS:
NUMBER OF FEMALES:
Pared
Falled to mate
Inseminated
Not pregnant
Mistimed pregnancy
Pregnant
PRE - COITAL INTERVAL - DAYS
MEAN
SD
N
(COPULATION INDEX (%)
PREGNANCY RATE (%)
FERTILITY INDEX (%)
Caesarean phase (Inseminated females)
- With vlable fetuses
Lactation phase (Inseminated females)
- Females with live puns (2)
- Euthanized moribund post-partum
- Total litter death post-partum
- Reared pups to wearing
GESTATION INDEX (%)
44
(b) (4)
43
3.0
21
22
100
C: Caesarean phase
L: Lactation phase
(' mistimed pregnancy for one par of rats
(b) (4)
© Including one euthanized mortbund post-partum female from group (b) (4)
44
O
A4
1C+1L
O
42
2.8
§ §
21
21
D
21
100
101

BLA 125742
Table 68: Summary of cohabitation data and maternal performance in littering and Caesarean subsets
Caesarean data
Gravid uterus weight
No test article-related effects on mean gravid uterus weight were reported.
Mean gravid uterus weight and maternal body weight change
Devist G21 Relative to Mating (Liller: A]
Sex: Femele
Control
Orag
ENT16261 (b) (4)
ENT16262
30m.g
ENT16263 (b) (4)
Gresid
Uterus (g)
Necropsy
EW (g)
Adiusted
EW (g)
NetaWc
from Ge [a)
Mess
SO
N
SO#
Mess so
N
5DP
Mes-
SO
N
§O#
Mess so
N
86.32 R.K
7.69
21
(b) (4)
AT FE
13.48
(b) (4)
386.51 121
24.72
21
1.5
35147
25.24
21
-11
280.19 L*
22.08
21
15.75
104.25
1.27
21
NetBIC
-Uterine Wt [g)
Mesn Fcetal
WE B08)
Mes-
SO
N
§O#
Mess
SO
17.93
7.54
21
4.B9
▶
023
21
No. Live
Foeluse:
Mess
SO
§D#
13.2 R.K°
1.6
-5.B4
93.20 dd
15.12
21
1061
5.55ddd
8.55
21
-69.DE
4.90
0.30
2
Des
131
21
-0.4
+ [Footnote is displayed in the comments and markers page]
1 [R,k - Automatic transformation: Rank, (all groups) test: Kruskal-Wallis p < 0.05]
2 Id - Test: Dunnett Non- Parametric 2-sided p < 0.051
3 [I,a - Automatic transformation: Identity (no transformation), (All groups) Test: Analysis of variance p < 0.057
4 [L - Automatic transformation: Log]
5 [R,kkk - Automatic transformation: Rank, (all groups) Test: Kruskal-Wallis p < 0.001]
6 Idd - Test: Dunnett Non- Parametric 2-sided p < 0.01]
7 Iddd - Test: Dunnett Non- Parametric 2-sided p < 0.0011
8 [I,aaa - Automatic transformation: Identity (no transformation), (all groups) test: Analysis of variance p < 0.0011
9 [ddd - Test: Dunnett 2-sided p < 0.0 1]
102

BLA 125742
O [d - Test: Dunnett 2-sided p < 0.05]
Table 69: Mean gravid uterus weight and maternal body weight change
Pregnancy incidence
No test article-related effects on pregnancy incidence were reported. At the terminal Caesarean examinations, there were 21/22, (b) (4), 21/22, and (b) (4) pregnant/mated females in groups 1,%, 3, and , respectively. All of which had viable fetuses.
Pre-implantation data
No test article-related effects on the pre-implantation data were reported. The mean numbers of corpora lutea and implantation sites were comparable in all groups.
The mean percentage pre-implantation loss was higher in groups 3 (b) (4) (9.77% and (5) (4), respectively) compared with the control group (4.09%). However, the differences remained within the historical control data range ((b) (4) ) for pivotal studies. Thus, the difference
was considered to be incidental.
Post-implantation data
No test article-related effects on embryo-fetal survival were reported. The mean percentage post-implantation loss and the mean live litter size were comparable in all groups and consistent with the historical control data.
Fetal data
No test article-related effects on mean fetal weight or fetal sex ratio were reported.
Mean Caesarean section data
Sex: Female
Control
Omcg
BNT16261 (b) (4)
BNT162b2
30mcg
BNT16263 (b) (4)
Day(s) Relative to Mating (Litter: A)
Females Pregnant [CHSQFS]
Dams with Viable Foetuses
No. of Corpora Lutea [GEN AN]
N+ve
21
(b) (4)
21
(b) (4)
No. of Implantations [GEN AN]
Pre-Implantation Loss IGEN ANI
Pre-Implantation Loss (%) [KWLWCX]
No. of Early Resorptions [GEN AN]
Early Resorptions (%) IKWLWCXI
No. of Late Resorptions (GEN ANI
Mean
SD
Sum
Mean
SD
Sum
Mean
SD
Sum
Mean
SD
Mean
SD
Sum
Mean
SD
Mean
SD
21
14.7 |1
1.6
309
14 1
R2
1.6
296
R2
0.6 R.k3
1.0
13 R.k3
4.09 k5
6.56
0.8
R2
1.2
16
R2
5.04
7.23
01 R2
0.4
21
15.5
2.1
326
14 0
2.2
294
1 5
d4
1.3
32
d4
9.77 dA
8.09
0.7
1.0
14
4.62
6.12
0.2
0.5
103

BLA 125742
Sex: Female
Day(s) Relative to Mating (Litter: A)
Late Resorptions (%) KWLWCX]
No. of Dead Foetuses GEN ANI
Post-Implantation Loss [GEN AN]
Post-Implantation Loss (%) [KWLWCX]
No. of Live Foetuses [GEN ANI
No. of Male Foetuses [GEN AN]
No. of Female Foetuses [GEN ANI
Male Foetuses (%) [KWLWCX]
Total Litter Weight (g) [GEN ANI
Mean Foetal Weight (both) (g) [GEN AN]
Mean Foetal Weight (M) (g) [GEN AN]
Mean Foetal Weight (F) (g) [GEN AN]
Sum
Mean
SD
Mean
SD
Sum
Mean
SD
Sum
Mean
SD
Mean
SD
Sum
Mean
SD
Sum
Mean
SD
Sum
Mean
SD
Mean
SD
% Diff
Mean
SD
%Diff
Mean
SD
Mean
SD
Control
Omcg
3
R2
1.05
2.66
0.0 R2
0.0
R2
09 R2
1.2
19
R2
6.10
7.64
132 R.KI
1.6
277 R,k'
6.1
1.7
129
2
7.0
12
2.1
148
46.96
14.27
64.23
5.91
21
4 89
0.23
21
5 00 12
0.21
4.79 |2
0 24
[KWLWCX] - Kruskal Wallis & Wilcoxon
[GEN ANI - Generalised Anova/Ancova Test
1 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]
2 [1 - Automatic Transformation: Identity (No Transformation)]
3 IR.kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.011
4 Id - Test: Dunnett Non-Parametric 2 Sided p < 0.051
Table 70: Mean Caesarean section data
BNT162b1 (b) (4)
(b) (4)
BNT162b2
30mcg
4
1.23
3.27
0.0
0.0
0
0.9
1.2
18
5.85
7.28
13.1
2.1
276
§ 7
2.0
141
6.4
1.5
135
50.66
10.69
64.32
10.53
21
0.14
4.90
0.30
21
0.25
5.02
0.30
4 77
0.32
BNT162b3 (b) (4)
(b) (4)
104

BLA 125742
Fetal examinations
The numbers of fetuses (litters) submitted to the different examinations were as follows:
Group No.
1
4
External examination
2
3
1277 (21) (b) (4) 276 (21) (b) (4)
Internal (visceral) examination (body)
Fixed head examination
Skeletal examination (head and body)
133 (21)
132 (21)
133 (21)
132 (21)
144 (21)
144(21)
No test article-related effects on fetal morphology were reported. This is consistent with no corresponding malformations in pups.
External observations
No test article-related effects on fetal external morphology were reported. (b) (4)
In group 3, one fetus had gastroschisis and one fetus had a small mouth and agnathia. These malformations are part of the background data for this strain of rat () (4): WI(Han)) and were considered incidental in view of their isolated and sporadic nature.
Visceral observations
No test article-related effects on fetal soft tissue morphology were reported. (b) (4)
(b) (4)
One fetus of group 3 was reported with a right-sided aortic arch b) (4)
These findings are also part of the background of
findings for this strain of rat (b) (4) : WI(Han)) and were considered incidental in view of their isolated incidences.
The other less severe soft tissue anomalies and variations are part of the background data for this strain of rat and were also incidental.
38 Kuwagata et al. Historical control data on developmental toxicity studies in rats. Congenital anomalies. 2018 59, 125-131.
105

BLA 125742
Skeletal observations
No test article-related effects on fetal skeletal morphology were reported. b) (4)
. One fetus from group
3 had short and fused mandibles. These malformations associated with the abnormalities reported externally and were considered incidental in view of their isolated incidences.
As part of the background data for this strain of rat (and were considered incidental), other less severe skeletal anomalies and variations, such as supernumerary lumbar ribs, 7 lumbar vertebrae or incomplete ossification of thoracic centrum were reported.
Summary of Foetal External, Visceral and Skeletal Observations
Exam Type: Visceral Body (Rat)
Heart
Heart, Ventricular septum defect - (M)
Liver
Liver, Abnormal lobation - (A)
Lung
Lobe, Absent - (A)
Lobe, Supernumerary - (A)
Major blood vessel
Aortic arch, Right-sided - (M)
Ductus arteriosus, Narrowed - (M)
Subclavian artery, Malpositioned - (A)
Umbilical artery, Transposed - (V)
Exam Type: Skeletal Head (Rat-G21)
Skull
Cranium, Acrania - (M)
Hyoid, Incomplete ossification - (A)
Interparietal, Incomplete ossification - (V)
Mandible, Fused - (M)
Mandible, Misshapen - (A)
Mandible, Short - (M)
Parietal, Incomplete ossification - (V)
Presphenoid, Incomplete ossification - (A)
Squamosal. Incomplete ossification - (V
Supraoccipital, Incomplete ossification - (V)
Number of Fetuses Examined:
Number of Litters Examined:
Fetuses N%)
Litters N/9)
Litters N(%)
Fetuses N%)
iters NIS
Fetuses N%)
Litters N/%
Litters N%)
Fetuses N%)
Litters N%)
Fetuses N%)
Litters N%)
Fetuses N1%)
Litters N%)
Number of Fetuses Examined:
Number of her Fyamined
Fetuses N/9)
Fetuses N%)
Litters N%)
Fetuses N(%)
Litters N/96)
Fetuses N%)
Litters N/%)
Litters N(%)
Fetuses N%
ters NIC
Fetuses N(%)
Litters N(%)
Fetuses N/C
Litters N%)
Fetuses N%)
Litters N/%
1 [c - Group Factor Chi-Squared & Fisher's Exact Test: Chi-Squared p < 0.05]
Control
21
00.0)
00.0)
10.8)
1148)
00.0)
00.0)
00.0)
00.0)
00.0)
00.0)
00.0)
00.0)
00 O
00.0)
753
6(28.6)
Control
21
00.0)
00.0)
010.0)
00.0)
32.1)
3114 31
00.0)
010.0)
00.0)
00.0)
00.0)
00.0)
00.0)
00.0)
10.7
14.8
00.0)
00.0)
00.0)
00.0)
BNT162b' (b) (4)
21
010.0)
010.0)
010.0)
010.0)
10.8)
1(4.8)
0(0.0)
010.0)
10.8)
1(4.8)
010.0)
00.0)
010.0)
00.0)
139.8
838.1)
BN716262
21
107
3/14 31
10.7)
10.7
312.1)
3/14 31
295
106

BLA 125742
[Exam Type: Skeletal Body (Rat-G21)
Vertebra, Presacral vertebral arches = 27 - (A)
Forepar
Phalanx, Unossified - (A)
Hindpaw
Metatarsal, Unossified, 1st digit - (V)
Phalanx, Unossified, proximal 2nd to 5th digits - (V)
Ribs
Ribs Subemumerary cervcal- (A)
Ribs, Superumerary lumbar - (A)
Ribs. Thick - (A)
Ribs, Wavy - (A)
Ribs, Supemumerary lumbar, short - (V)
Exam Type: Skeletal Body (Rat-G21)
Ribs (Continued...)
Ribs, Superumerary lumbar, short - (V)
Sternebra
Sternebra, Asymmetric - (A)
Sternebra. Extra ossification site - (A)
Sternebra, Incomplete ossification, 1st/3rd - (A)
Sternebra. Incomplete ossification. 2nd/4th - )
Sternebra, Incomplete ossification, 6th - (V)
Sternebra, Minor fusion - (A)
Sternebra, Misshapen - (A)
Sternebra, Unossified. 5th - (A)
Vertebra
Caudal, Number < 5 - (A)
Exam Type: Skeletal Body (Rat-G21)
Vertebra (Continued ...)
Caudal, Number < 5 - (A)
Cervical Fused arch - (A)
Cervical Incomplete ossification of arh. (A)
Cervical, Multiple abnormalities - (M)
Cervical. Odontoid process unossified - M
Cervical Unossied centrum- (V
Lumbar, Number = 7 - (A)
Sacral, Misshapen arch - (A)
Thoracic, Bipartite ossification of centrum - (A)
Thoracic, Incomplete ossification of centrum, 1st to 9th - (A)
Thoracic Incomnlate occ rfeation of centrum ith to 13th - (A
Number of Fetuses Examined:
Number of her Eyamined
Fetuses N%)
Litters N%)
Litters N%)
Fetuses N%)
Number of Fetuses Examined:
Number of litters Examined
Litters N(%)
Litters N%)
Fetuses N/%)
Litters N%)
Fetuses N/°)
Litters N(%)
Fetuses N/%)
Litters N%)
Fetuses N%)
Litters N%)
Fetuses N/°)
Litters N%)
Fetuses N/°)
Litters N(%)
Fetuses N/%)
Litters N%)
Fetuses N/%)
Number of Fetuses Examined
Number of Litters Examined:
Litters N%)
Fetuses N/96)
Litters NI°
Fetuses N/%)
Litters N%)
Fetuses N/%)
Liters N/°)
Fetuses N%)
itero NIChI
1 more NIC
Litters N%)
Fetuses N(%)
Litters N(%)
Fetuses N/%)
Fetuses N/%)
Control
21
00.0)
00.0)
96.3)
763.3
322.1)
3(14.3)
46(31.9
11(52.4)
32.1)
3(14.3)
3(2.1)
3(14.3)
2(1.4)
148
0(0.0)
00.0)
57339.6
Contro
21
1781.0)
107
114 8)
00.0)
010.0)
10.7
148
10.7)
1(4.8)
010.0)
00.0)
10.7
14.8)
00.0)
00.0)
00.0)
00.0)
00.0)
Conterl
21
00.0
00.0
00.0
00.01
00.0)
00.0)
00.0
96.3
7633
312.1)
3(14.3
107
14.8)
00.0
010.01
00.0
00.0
10.7
14.81
6/4.2)
BNT1626
-(b) (4)
UT169
21
10.7
14.8)
6142
3/143)
312.1)
3114.3)
22(15.3)
733.3)
010.0)
010.01
128.31
6(28.6)
4¢2.8)
314 31
10.7
1(4.8)
71(49.3)
BNT16264
21
1885.7)
00.0
010.0)
010.0)
010.0
10.7
148
2/1.4)
29.5
00.01
010.0)
0,0.0)
010.0)
0,0.0)
010.0)
010.0)
010.0)
211.4)
BNT1626
21
29.5
010.0)
00.0)
211.4)
29.5
010.0)
010.0)
614.2
4(19.0)
211.4)
295
312.1)
295
00.0
010.0
00.0
010.0
32.1
3(14.3)
96.31
BNT162b:
_ (b) (4)
107

BLA 125742
Exam Type: Skeletal Body (Rat-G21)
Control
Number of Fetuses Fyamined
Number of Litters examinec
21
vertebra continued..
Thoracic, Incomplete ossification of centrum, 10th to 13th. - (A)
Thoracic, Multiple abnormalities - (M)
Thoracic, Number = 14 - (A)
LLitters N(%)
Fetuses N(%]
Litters N(%)
Fetuses N(°1
Litters N(%)
523.8) c'
0(0.0)
010.0)
010.0)
0(0.0)
Table 71: Summary of Foetal External, Visceral and Skeletal Observations Delivery and litter data
7615265
BNT16262
21
9/42.9)
010.0)
010.0)
010.0)
010.0)
Parturition and gestation length
No test article-related effects on parturition and gestation length were reported. In groups 1,, 3, and, there were 22, o 1,21 and (' females that completed delivery and had liveborn pups giving a gestation index of 100%, (b) (4), 100% and (b) (4), respectively. This was consistent with the background data for this strain of rat.
In all groups, the mean duration of gestation (approximately 22 days) was comparable. (b) (4)
Pre-Birth Loss
The mean percentage pre-birth loss was higher in group (b) (4) and group (b) (4) when compared with the control group (6.8%). However, the value remained consistent with the historical control data range (from (b) (4) ) for pivotal studies. Thus, the difference was considered to be incidental.
Consequently, the mean number of pups delivered was lower in groups (b) (4) respectively) compared with the control group (13.3). However, the values remained consistent with the historical control data range (from (b) (4) ) for pivotal studies.
Pup Viability and Litter Sizes (b) (4)
108

BLA 125742
(b) (4)
Sex: Female
Days) Relative to Littering (Litter: A)
Females Completing Delivery [CHSQFS]
with Liveborn Pups [CHSQFS] with Stillborn Pups [CHSQFS]
with all Stillborn Pups [CHSQFS] with all Dead PND 21 [CHSQFSI
Gestation Length (Days) [GEN AN]
Number of Implantation Sites [GEN AN]
Pre-Birth Loss (%) [GEN AN]
Pups Delivered/Litter [GEN ANI
Live Pups PND 0 [GEN ANI
Live Pups PND 1 [GEN AN]
Live Pups Precull [GEN ANI
Live Pups Postcull [GEN ANT
Live Pups PND 7 [GEN ANI
Live Pups PND 10 [GEN ANI
Live Pups PND 14 [GEN ANI
N+ve
N+ve
N+ve
N+ve
N+ve
Mean
SD
N
Mean
SD
N
Sum
Mean
SD
N
Mean
SD
N
Sum
Mean
SD
N
Sum
Mean
SD
N
Sum
Mean
SD
N
Sum
Mean
SD
N
Sum
Mean
SD
N
Sum
Mean
SD
N
Sum
Mean
SD
N
Control
Omcg
22
22
3
0
0
22.1
0.4
22
14.3 13
2.2
22
314
13
6.80 R, k4
8 75
22
13.3 R.k4
25
22
293 R.k4
13.0 R.k'
25
22
287 R.kI
13.0 R.KI
2.4
22
785 R.kl
12.9 R.kI
23
0.0
22
176
R3
8.0 R3
0.0
22
176
R3
80 RI
0.0
22
176
RI
8.0 RI
00
22
BNT162b1 (b) (4)
BNT16262
30mcg
21
21
2
0
22.0
0.7
21
14.2
2.2
21
298
8.22
15 51
21
13.1
3.1
21
276
13.0
31
21
274
13.0
3.0
21
273
12.9
2.9
21
271
7.8
1 1
21
163
78
1.1
21
163
7.8
1.1
21
163
7.8
1.1
21
BNT162b3 (b) (4)
109

BLA 125742
Sex: Female
Day(s) Relative to Littering (Litter: A)
Control
Omcg
BNT162b1 (b) (4)
BNT162b2
30mcg
BNT162b3 (b) (4)
Live Pups PND 17 [GEN AN]
Live Pups PND 21 [GEN ANI
Sum
Mean
SD
N
Sum
Mean
SD
N
Sum
Sum
Sum
Sum
Sum
Mean
176 RI
80 RI
0.0
22
176
RI
8.0 RI
0.2
22
175
6
3
§ a
98.0
99 0
99 4
51.0
49.7
RI
163
7.8
1.1
21
163
7.8
1.1
21
163
2
3
0
5
99.3
98.9
100.0
48.0
47.6
[GEN ANT - Generalised Anova/Ancova Test
2 [dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01]
4 (R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]
Dead, Miss., Cannib. PND 0 [CHSOFS]
Dead, Miss., Cannib. PND 1-4 [CHSQFS]
Dead, Miss., Cannib. PND 5-21 [CHSQFS]
Dead, Miss., Cannib. PND 0-21 [CHSQFS]
Live Birth Index (%)
Viability Index (PND 0-4) (%)
Weaning Index (PND 4-21) (%)
Sex Ratio PND 1 - % Males [CHSQFS]
Sex Ratio PND 21 - % Males [CHSQFS]
Mean
[CHSQFS] - Chi-Squared & Fisher's Exact
1 (R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01]
3 [I - Automatic Transformation: Identity (No Transformation)]
5 [d - Test: Dunnett Non-Parametric 2 Sided p < 0.05]
Table 72: Delivery and litter data
Pup Clinical Observations
No test article-related effects on pup clinical observations or external abnormalities were reported.
110

BLA 125742
Pup Weights
No test article-related effects on mean pup weight throughout the pre-weaning period were reported.
(b) (4)
111

BLA 125742
Mean pup body weight (grams)
Sex: Female
Day(s) Relative to Littering (Litter: A)
Mean Pup BW - Males d1 [GEN AN]
Mean Pup BW - Males d4 [GEN ANT
Mean Pup BW - Males d7 [GEN ANT
Mean Pup BW - Males d10 [GEN AN]
Mean Pup BW - Males d14 [GEN ANI
Mean Pup BW - Males d17 [GEN ANI
Mean Pup BW - Males d21 [GEN ANI
Mean Pup BW - Males d4 Postculing [GEN AN]
Mean Pup BW - Females d1 (GEN AN]
Mean Pup BW - Females d4 [GEN ANI
Mean Pup BW - Females d7 [GEN AN]
Mean Pup BW - Females d10 (GEN AN]
Mean
SD
%Diff
Mean
N
%Diff
Mean
N
%Diff
Mean
N
%Diff
Mean
SD
N
%Diff
Mean
N
% Diff
Mean
N
%Diff
Mean
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
Control
Omcg
6.25 RI
0.82
22
9.71 12
1.26
22
16.14 RI
1.76
22
23.79 RI
2.17
22
34.35 12
2.76
22
41.64 |1
3.10
22
55.53 1
4 02
22
971 |1
1.31
22
6.00 |1
0.82
22
9 47 |1
1.25
22
15.77 RI
1 72
22
23.35 RI
2.21
22
BNT162b1
(b) (4)
BNT16262
30mcg
6.27
0.73
20
0.23
9.81
1.21
20
1.00
16.47
1.74
20
2.07
24.24
1.87
20
1.87
34.93
2.13
20
1.69
42.07
2.36
20
1.04
56.10
3.22
20
1 03
9.78
1.24
20
0.66
6.06
0.73
21
0.97
9 58
1 33
21
1 25
16 10
1 75
21
2.14
23.82
1.85
21
BNT162b3
(b) (4)
112

BLA 125742
Sex: Female
Day(s) Relative to Littering (Litter: A)
Mean Pup BW - Females d14 [GEN AN]
Mean Pup BW - Females d17 [GEN ANI
Mean Pup BW - Females d21 [GEN AN]
Mean Pup BW - Females d4 Postculling [GEN AN]
Mean Pup Body Weight d1 [GEN AN]
Mean Pup Body Weight d4 [GEN AN]
Mean Pup Body Weight d7 [GEN AN]
Mean Pup Body Weight d10 IGEN AN]
Mean Pup Body Weight d14 [GEN AN]
Mean Pup Body Weight d17 [GEN AN]
Mean Pup Body Weight d21 [GEN AN]
Mean Pup BW d4 Postculling [GEN AN]
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
%Diff
Mean
SD
N
Control
Omcg
33.71 12
2.88
22
40.69 12
3 16
22
54.02 12
4 18
22
9.49 |1
1 25
22
6.13 R2
0.82
22
9.60 11
1.25
22
15.95 R2
1 71
22
23.57 R2
2.15
22
34.03 |1
2.78
22
41 16 |1
3.11
22
54.75 11
4.07
22
9.60 |1
1.26
22
BNT162b1
30mcg
2.73
33.91
BNT162b2
(b) (4)
BNT162b3
(b) (4)
86⅜⅛⅜⅜8£=4388⅔88⅜8⅜93888838⅜⅜8⅜⅜¾¾½838⅝88¾
16.94 S3
113

BLA 125742
Sex: Female
Day(s) Relative to Littering (Litter: A)
Control
Omcg
BNT162b1 (b) (4)
(b) (4)
BNT162b2
30mcg
1.51
BNT162b3 (b) (4)
(b) (4)
%Diff
Table 73: Mean pup body weight (grams)
Pup Physical and Functional Development
No test article-related effects on pre-weaning physical (pinna unfolding and eye opening) and functional (pupil and auditory reflexes) development were reported
Summary of reflex and physical development
Group
Dose level
1
Control
0 ug
ク
BNT162b1 (b) (4)
3
BNT16262
BNT16263
30ug
-(b) (4)
PINNA UNFOLDING
- % of pups positive:
day 1 post-partum day 2 post-partum dav 3 post-partum day 4 post-partum
5
51
98
100
6
51
99
100 (3)
EYE OPENING
- % of pups positive:
day 12 post-partum day 13 post-partum day 14 post-partum dav 15 post-partum day 16 post-partum day 17 post-partum
3
19
83
9
79
99
100
96
100
PUPILLARY REFLEX - day 21 post-partum
- % of pups positive:
AUDITORY REFLEX - dav 21 post-partum
- % of pups positive:
100
100
100
100
(2) values excluded for three pups that were not observed after PND14 in error
(3). 99.7%. one unselected pup for culling was not observed after PND4
* p 5 0.05; *** p = 0.001
Table 74: Summary of reflex and physical development
Pup Necropsy Findings
No test article-related effects on pup macroscopic observations or malformations were reported.
Necropsy Findings of Adult Females
Test article-related macroscopic findings were reported at the injection sites (firm area, enlarged, edematous area and/or pale). These findings were consistent with the administration of the vaccine and an inflammatory/immune response localized to the injection site.
114

BLA 125742
Across all groups (including controls), abnormalities of the liver (diaphragmatic hernia, mottled
surface, abnormal shape or adherent mass) were reported for isolated females and were
considered incidental.
Across all groups (including controls), alopecia and/or sores/crusts were also reported for isolated females and were considered incidental
Summary of maternal macroscopic observations
Removal Reason: TERMINAL SACRIFICE
--- FEMALES
Control
BNT162b1 BNI162b2 BNI162b
Omee (b) (4)
30meg
43
(b) (4)
Number of Animals on Scuov:
Number of Animals Completed:
LIVER;
submiccea..........
No Visible Lesions........
Hernia; diaphracm: between right and left median lobeg
Mottled surface:
lobes
Abnormal shave: left median lobe
smalli lerc median ope ..................... .....
Mass a: acherent to surroundino vassue,
......''................
papillary process; solid; dark; heterogeneou
IDENTIFICATION;
Suomitteo............
No Visible Lesions.
SKIN/SUBCUTIS;
submicce.........
No Visible Lesions....
AlODecia: sinGle;
forelimi;
r1cht
ginae: forelimo: lett
Alopecia: single: abdominal region: choracic
Alopecia: single; choracic region
Alopecia; single; choracic region;
cioco)
Alovecia: richt: forepaw:
abdominal
left
Sore
Sore crust: manv: forelimo: left
Sore/crust; single; right
Sore/ennes: sinde:
Forsimo:
Sore crust: single: hindimo:
етс
Sore/crust: single: abdominal regior
NO CORRELATE;
Smomi teed.
NO CORRELATE; (continued)
No Visible Lesions.
No correlate
INJECTION SITE 1;
Submitted..........
No Visible Lesions
Dale
INJECTION SITE 2:
Suomictea........
No visible Lesions
Firm area
Enlarged
Oedematous area
Pale ........
NO CORRELATE;
Submitted.........
No rasiole lesione
No conrelare
LIVER;
Stomitted
No
Visible Lesions
Pale: al obss
SPLEEN;
No Visible Lesions.
Enlarged
IDENTIFICATION:
Submitted..........
No Visible Lesions.
SKIN/SUBCUTIS;
Submitted..........
No Visible Lesions................
Alonecia: sinale: forelim:
abdominal region:
(9)
(5)
000008
Table 75: Summary of maternal macroscopic observations
115

BLA 125742
Summary (b) (4)
, BNT16262 (b) (4) ) resulted in clinical signs and macroscopic
findings localized to the injection site as well as transient body weight and food consumption effects after each dose administration. These maternal findings might be related to the administration of the vaccine and an inflammatory/immune response.
No test article-related effects on estrous cycles, pre-coital interval, mating, fertility and pregnancy index, or on any ovarian, uterine, or litter parameters, including F1 survival, growth, external, visceral, and skeletal morphology, or effects on pre-weaning physical and functional development of the F1 pups were reported.
Four doses (2 prior to mating and 2 during gestation) administration of the test articles (b) (4)
, BNT16262, (b) (4) )elicited SARS-CoV-2 neutralizing antibody responses in the majority of females just prior to mating (M-14), at the end of gestation (GD21), and at the end of lactation (LD21). Also, SARS-CoV-2 neutralizing titers were detected in most offspring (fetuses on GD21 and pups on PND21). Prior to vaccine administration or in saline-administered control animals, SARS-CoV-2 neutralizing antibody titers were not reported.
Conclusion
Test article-related effects on body weight, food consumption, and effects localized to the injection site after each dose administration were reported. No test article-related effects on mating performance or fertility in FO female rats or on embryo-fetal or postnatal survival, growth, or development of the F1 offspring were reported.
Test article-related immune responses were confirmed in FO female rats following administration of each vaccine candidate and these responses were also detectable in the F1 offspring (fetuses and pups).
116

6 pages have been determined to be not releasable: (b)(4)

BLA 125742
(b) (4)
For complete historical data, please visit appendix 29 on page 1084 of the study report submitted in amendment number 165.
References:
1. Habibzadeh P, Stoneman EK. The Novel Coronavirus: A Bird's Eye View. Int J Occup Environ Med. 2020; 11 (2): 65-71.
2. Vogel AB, Lambert L, Kinnear E, Busse D, Erbar S, Reuter KC, et al. Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses. Mol Ther. 2018; 26 (2): 446-55.
3. Moyo N, Vogel AB, Buus S, Erbar S, Wee EG, Sahin U, et al. Efficient Induction of T Cells against Conserved HIV-1 Regions by Mosaic Vaccines Delivered as Self-Amplifying mRNA. Mol Ther Methods Clin Dev. 2018; 12: 32-46.
4. Pardi N, Hogan MJ, Pele RS, Muramatsu H, Andersen H, DeMaso CR, et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature. 2017; 543 (7644): 248-51.
5. Colombo S, Buclin I, Décosterd LA, Telenti A, Furrer H, Lee BL, Biollaz J, Eap CB (October 2006). "Orosomucoid (alphal-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation". Clinical Pharmacology and Therapeutics. 80 (4): 307-18. do: 10.1016/j.clpt.2006.06.006.PMID17015049.
6. Urien S, Brée F, Testa B, Tillement JP (November 1991). "pH-dependency of basic ligand binding to alpha 1-acid glycoprotein (orosomucoid)". The Biochemical Journal. 280 ( Pt 1) (1): 277-80. PMC 1130632. PMID 1741754.
7. Zimmermann-Belsing T, Rasmussen AK, Feldt-Rasmussen U, Bog-Hansen TC (February 2002). "The influence of alphal-acid glycoprotein (orosomucoid) and its glycoforms on the function of human thyrocytes and CHO cells transfected with the human TSH receptor". Molecular and Cellular Endocrinology. 188 (1-2): 241-51. do: 10.1016/0303-7207(01)00650-5. PMID 11911961.
123

BLA 125742
8. Dietrich JW, Landgrafe G, Fotiadou EH (2012). "TSH and thyrotrophic agonists: key factors in thyroid homeostasis". Journal of Thyroid Research. 2012: 351864. do: 10.1155/2012/351864. PMC 3544290. PMID 23365787.
9. Fischer K, Kettunen J, Würtz P, Haller T, Havulinna AS, Kangas AJ, Soininen P, Esko T, Tammesoo ML, Mägi R, Smit S, Palotie A, Ripatti S, Salomaa V, Ala-Korpela M, Perola M, Metspalu A (February 2014). "Biomarker profiling by nuclear magnetic resonance spectroscopy for the prediction of all-cause mortality: an observational study of 17,345 persons". PLoS Medicine. 11 (2): e1001606. do: 10.1371/journal.pmed. 1001606. PMC 3934819. PMID 24586121.
10. Ulich TR, del Castillo J, Souza L. Kinetics and mechanisms of recombinant human granulocyte-colony stimulating factor-induced neutrophilia. Am J Pathol 1988;133 (3): 630-38.
11. Abreu R, Quinn F, Giri PK. Role of the hepcidin-ferroportin axis in pathogen-mediated intracellular iron sequestration in human phagocytic cells. Blood Adv 2018;2(10): 1089-100.
12. Brooks MB, Turk JR, Guerrero A, et al. Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability. PLoS One
2017;2(1),0169976.
13. Kim A, Fung E, Parikh SG, et al. A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin. Blood 2014;123(8): 1129-136.
14. Wrighting DM and Andrews NC. Interleukin-6 induces hepcidin expression through STAT3. Blood 2006;108(9): 3204-09.
15. Ivens IA, Achanzar W, Baumann A, et al. PEGylated biopharmaceuticals: current experience and considerations for nonclinical development. Toxicol Pathol 2015 Oct; 43(7):959-83.
124