BioNTech SE
An der Goldgrube 1255131 Mainz, Germany
Phone: +49 (0)6131 9084-0
Telefax: +49 (0)6131 9084-390

090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)R&D STUDY REPORT No. R-20-0072

EXPRESSION OF LUCIFERASE-ENCODING
MODRNA AFTER I.M. APPLICATION OF GMPREADY ACUITAS LIPID NANOPARTICLE
FORMULATION

Version 03
Date: 27 Nov 2020
Reported by(b) (6)

Test item: modRNA encoding luciferase
Key words: COVID-19, modRNA, biodistribution, mouse, bioluminescence assay
This R&D report consists of 36 pages.

Confidentiality Statement: The information contained in this document is the property and copyright of
BioNTech RNA Pharmaceuticals GmbH. Therefore, this document is provided in confidence to the recipient (e.g.,
regulatory authorities, IECs/IRBs, investigators, auditors, inspectors). No information contained herein shall be
published, disclosed, or reproduced without prior written approval of the proprietors.

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090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)TABLE OF CONTENTSTABLE OF CONTENTS ........................................................................................ 2
LIST OF FIGURES ................................................................................................ 3
LIST OF TABLES .................................................................................................. 3
LIST OF ABBREVIATIONS ................................................................................... 4
RESPONSIBILITIES .............................................................................................. 51
SUMMARY ........................................................................................... 62
GENERAL INFORMATION .................................................................. 8
2.1
Sponsor and Test Facilities .................................................................. 8
2.2
Participating Personnel ......................................................................... 8
2.3
Study Dates .......................................................................................... 8
2.4
Guidelines and Regulations .................................................................. 9
2.5
Changes and Deviations ....................................................................... 9
2.6
Documentation and Archive .................................................................. 9
3
INTRODUCTION ................................................................................ 10
3.1
Background ........................................................................................ 10
3.2
Objectives ........................................................................................... 10
3.3
Study Design ...................................................................................... 114
MATERIALS AND METHODS ............................................................ 12
4.1
Test Item ............................................................................................. 12
4.2
Control Item ........................................................................................ 12
4.3
Test System ........................................................................................ 12
4.4
Materials ............................................................................................. 13
4.5
Methods .............................................................................................. 14
4.5.1
Animal Care ........................................................................................ 14
4.5.2
Animal Monitoring ............................................................................... 15
4.5.3
Endpoint of Experiment / Termination Criteria .................................... 15
4.5.4
Injection of Test and Control Items ..................................................... 15
4.5.5
Bioluminescence Measurements ........................................................ 16
4.5.6
Blood Sampling via the Vena Facialis................................................. 16
4.5.7
Luminex-based Multiplex Assay (ProcartaPlex Multiplex Immunoassay)............................................................................................................ 16
4.5.8
Luciferase-specific ELISA ................................................................... 17
4.5.9
ELISpot Analysis................................................................................. 17
4.5.10Statistical Analysis .............................................................................. 18
5
RESULTS ........................................................................................... 19
5.1
Bioluminescence Measurements ........................................................ 19
5.2
Liver Expression (b) (4) vs LNP8 ......................................................... 21
5.3
Luminex-based Multiplex Assay ......................................................... 21
5.4
Luciferase-specific ELISA ................................................................... 23
5.5
IFN- ELISpot Assay ........................................................................... 23
6
CONCLUSION .................................................................................... 25
7
DOCUMENT HISTORY ...................................................................... 26
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8
REFERENCES ................................................................................... 27
9APPENDIX ......................................................................................... 28
Appendix 1: Animal Observations ........................................................................ 28
Appendix 2: Certificates of Analysis .................................................................... 29
Appendix 3: Raw Data IFN- ELISpot .................................................................. 32
Appendix 4: Statistical Analysis ........................................................................... 33

LIST OF FIGURES

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Figure 1: Bioluminescence measurement using the LNP-formulated modRNA
encoding for luciferase ........................................................................................ 20
Figure 2: Bioluminescence measurement in the liver using the LNP-formulated
modRNA encoding for luciferase ......................................................................... 21
Figure 3: Activation of the innate immune system by LNP-formulated modRNA
encoding for luciferase ........................................................................................ 22
Figure 4: Luciferase-specific IgG ELISA on days -1 and 9 .................................. 23
Figure 5: ELISpot analysis using splenocytes on day 9 ....................................... 24

LIST OF TABLES
Table 1: Study design .......................................................................................... 11
Table 2: Lipid component formulations ................................................................ 12
Table 3: Materials ................................................................................................ 13
Table 4: Software ................................................................................................ 14
Table 5: Peptide pool for stimulation of splenocytes ............................................ 14

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LIST OF ABBREVIATIONS
ALC-0159

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ALC-0315
ALP
ANOVA
AUC
BALB/cBCIP
BNT162ConA
COVID-19
DPBS
ELISA
ELISpot
FELASA
GMP
HRP
IFN
IgG
IL
i.m.
IP-10
IVIS
LNP
Luc
MCP-1
MHC
MIP-1
modRNA
NBT
OD
p/s
SD
SOP
S proteinsaRNA
SARS-CoV-2
TNF

Proprietary PEG-lipid included as an excipient in the LNP formulation fromAcuitasProprietary amino-lipid included as an excipient in the LNP formulation fromAcuitasAlkaline phosphatase
Analysis of variance
Area under the curve
Mouse strain used in this study
5-Bromo-4-chloro-3-indolyl-phosphate
BioNTech’s vaccine program against COVID-19
Concanavalin A
Coronavirus disease 2019
Dulbecco’s phosphate-buffered saline
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent spot
Federation of European Laboratory Animal Science Associations
Good manufacturing practiceHorseradish peroxidase
Interferon
Immunoglobulin G
Interleukin
IntramuscularlyInterferon-gamma induced protein 10
In vivo imaging system
Lipid nanoparticle
Luciferase (from firefly Pyractomena lucifera)
Monocyte chemotactic protein 1
Major histocompatibility complex
Macrophage inflammatory protein 1
Nucleoside-modified mRNA
Nitro blue tetrazolium
Optical density
Photons per second
Standard deviation
Standard operating procedure
Spike protein
Self-amplifying mRNA
Severe acute respiratory syndrome coronavirus 2
Tumor necrosis factor

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SUMMARY

BioNTech is developing RNA-based vaccines designed to protect against the novel
coronavirus disease that emerged in 2019 (COVID-19). The BNT162 project involves
testing three RNA platforms, which are under development at BioNTech with thesurface or spike protein (S protein) of the novel coronavirus (SARS-CoV-2) as the viral
antigen. These RNAs will be formulated with a GMP-compatible LNP formulation
provided by Acuitas.

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In this study, the GMP-ready formulation containing the amino-lipid ALC-0315 and the
PEG-lipid ALC-0159 (in this report referred to as LNP 8 which is the identical(b) (4)composition as used in BNT162) was tested in comparison with a(b) (4) by Acuitas, (b) (4) and an in-house formulation, (b) (4)to characterize thebiodistribution of luciferase expressed by LNP-formulated nucleoside-modified mRNA
(modRNA). Activation of the innate immune system, formation of antibodies against
luciferase, and T-cell activation were also assessed.
Four groups of three BALB/c mice were injected intramuscularly (i.m.) with a total doseof 2 µg/animal of (b) (4) LNP8- or (b) (4) -formulated modRNA encoding luciferase
or with buffer (DPBS) as control. At 6 h, 24 h, 48 h, 72 h, 6 d, and 9 d after injection,
the in vivo luciferase expression was measured by luciferin application. Serumsamples were taken 1 day before and 6 h after immunization as well as on day 9 forquantification of the activation of the innate immune system in a Luminex-based
multiplex assay and antigen-binding antibody analysis in an IgG-specific ELISA.
Splenocytes were isolated on 9 d to assess the T-cell response by IFN- ELISpot
Assay.
An approximately 20-fold higher luciferase expression at the injections site wasobserved for modRNA-Luciferase (b) (4) and the GMP-ready modRNA-Luciferase
LNP8 when compared to modRNA formulated with (b) (4) The difference between
the area under the curve for (b) (4) and LNP8-formulated modRNA compared to buffer
control as well as to (b) (4) formulated modRNA was statistically significant. In
addition, luciferase expressed from the (b) (4) -formulated modRNA showed limited
drainage to the liver compared to LNP8-formulated modRNA.
A multiplex assay showed that the innate immune system was temporally activated by(b) (4) and LNP8-formulated modRNA. The activation was more pronounced for (b) (4)formulated modRNA than for modRNA formulated with LNP8, indicating a formulationrelated effect rather than a payload or expression level effect.
Treatment with modRNA with all tested LNP formulations did not induce the formationof luciferase-specific IgGs on day 9. However, a strong antigen-specific IFN- T-cell
response was measured by ELISpot assay on day 9 for (b) (4) and LNP8-formulated
modRNAs, with statistically significant differences between these test groups, thebuffer control, and the (b) (4) group.
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GENERAL INFORMATION

2.1

Sponsor and Test Facilities

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Sponsor
BioNTech RNA Pharmaceuticals GmbHAn der Goldgrube 1255131 Mainz
Germany

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Test Facility
BioNTech SE
An der Goldgrube 1255131 Mainz
Germany

2.2

Participating Personnel

Responsible person:(as defined in SOP-100-024)(b) (6)(b) (6)(b) (6)

BioNTech(b) (6)

Author:

BioNTech SE
Experimenter:
Immunization, blood sampling,
ELISA, ELISpot(b) (6)(b) (6)

BioNTech(b) (6)

Experimenter:
Luminex-based multiplex assay
BioNTech

2.3(b) (6)

Study Dates

Start of experiments:

14 JAN 2020

Completion of experiments:

23 JAN 2020

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Guidelines and Regulations

All experiments are executed in accordance with the existing standard operatingprocedures and described processes from BioNTech SE. Applicable documents arelisted below.










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2.5

Animal test application approval number: G18-12-007
SOP-030-071 Abtöten von Mäusen
SOP-030-072 Fixiergriff und Ohrmarkierung bei Mäusen
SOP-030-073 Betäubung bei Mäusen
SOP-030-074 Blutentnahme bei Mäusen
SOP-030-078 Isolierung muriner Splenozyten
SOP-030-079 Intramuskuläre Applikation bei Mäusen
SOP-030-110 IFN- ELISpot murin
SOP-090-013 Biological safety in laboratories

Changes and Deviations

Not applicable. There is no formal R&D plan available.

2.6

Documentation and Archive

Study reports are stored and archived according to SOP-100-003 Archiving of PaperBased Documents.
Raw data and evaluated data are saved at:

P:\BioNTechRNA\RN_R0030_AIRVAC\24_Preclinic\01_Vakzine Testing invivo Luc\IM#88 GMP ready LNP Acuitas modRNALab books:
o No. 1455 (complete study plan including results)
o No. 1835 (IVIS images and quantification, Luciferase ELISA,
ELISpot)o No. 1593 page 71-84 (Luminex-based multiplex assay)

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INTRODUCTION

3.1

Background

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In December 2019, an outbreak of pneumonia of unknown cause in Wuhan, Hubei
province in China started. The disease spread rapidly and in January 2020, the agent
was identified. By 1 April 2020, infection with the novel coronavirus (SARS-CoV-2) was
confirmed in approximately 820,000 people with more than 40,000 casualties1. A
vaccine is urgently needed and BioNTech decided to develop a rapid vaccine project(BNT162) with the surface or spike protein (S protein) of the virus as the viral antigen.

090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)The development of in vitro transcribed RNA as an active platform for the use ininfectious disease vaccines is based on the extensive knowledge of the company in
RNA technology, which has been gained over the last decade. The core innovation isbased on in vivo delivery of a pharmacologically optimized, antigen-coding RNA
vaccine to induce robust neutralizing Abs and accompanying/concomitant T-cell
responses to achieve protective immunization with minimal vaccine doses(Vogel et al. 2017, Moyo et al. 2018, Pardi et al. 2017).
At BioNTech, three different RNA platforms formulated with lipid nanoparticles (LNPs)
are under development, namely non-modified uridine-containing mRNA (uRNA),
nucleoside-modified mRNA (modRNA) and self-amplifying RNA (saRNA). In thepresent study, an LNP-formulated modRNA encoding luciferase was used
representatively to investigate the in vivo biodistribution and the immune response of
the vaccine candidates.
LNP formulations from a third party provider (Acuitas) were tested in comparison to the(b) (4)in-house formulation (b) (4)Acuitas(b) (4)(b) (4)

Acuitas also provided an LNP
formulation that is cGMP-ready, namely LNP8, which contains two proprietary lipids(ALC-0159 and ALC-0315) and has the identical composition as the LNP formulation
used in the BNT162 program.

3.2

Objectives

The objective of this study was to investigate the biodistribution of luciferase expressedby the LNP-formulated modRNA using bioluminescence measurements in BALB/cmice, as well as innate immune system activation, formation of antibodies against
luciferase and T-cell activation.

1 Coronavirus disease (COVID-2019) situation report 72, World Health Organization;/www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports

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Study Design

Four groups of three BALB/c mice were injected intramuscularly (i.m.) in the right andleft hind leg with each 1 µg of LNP-formulated modRNA encoding luciferase or withbuffer as control on day 0. At 6 h, 24 h, 48 h, 72 h, 6 d, and 9 d after injection, thein vivo luciferase expression was measured by luciferin application.
In addition, serum samples were collected on day -1 and 6 h and 9 d post immunizationand cytokine/chemokine level determination (multiple ) and on day 1 and 9 for
luciferase-specific ELISA. On day 9, spleens were resected for immunological analysis
using IFN- ELISpot assays.
Table 1: Study design

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Dose [µg/Formulation
mouse]

Treatmentschedule

End ofSample collection
experiment

Day 0

Day 9

Day 0

Day 9

2 µg
Acuitas proprietary Day 0(1 µg/leg)

Day 9

1

Buffer control N/A

2

modRNALuciferase(b) (4)
3

modRNALuciferase(b) (4)4

2 µg(1 µg/leg)N/A(b) (4)

modRNALuciferase
2 µg
ALC-0315:ALCLNP8 (GMP- (1 µg/leg) 0159:DSPC:Cholready)

Day 0

Serum ELISA on
days -1 and 9,
serum for Multiplex
assay on day -1, 6 h,
and 9 d;splenocytes for
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4

MATERIALS AND METHODS

4.1

Test Item

LNP-formulated modRNA encoding luciferase diluted to 0.05 mg/mL to obtaina dose of 1 µg in 20 µL application volume. For CoAs see Appendix 2:
Certificates of Analysis of RNA and LNPs.
Acuitas LNPs:(b) (4)oo LNP8 modRNA Luc, RNA-EH190611-01c, batch FM-1074-D, 90%
encapsulation, 1.0 mg/mL encapsulated RNA, diameter 71 nm,
polydispersity 0.053, storage temperature -80°C.

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BioNTech LNP:o(b) (4)

Table 2: Lipid component formulations

LipidsFormulation

Functional lipid 1

Functional lipid 2

Structural lipid 1

Structural lipid 2(b) (4)

Acuitasproprietary

Acuitasproprietary

Acuitasproprietary

Acuitasproprietary

LNP8

ALC-0315

ALC-0159

DSPC

Cholesterol(b) (4)(b) (4)(b) (4)(b) (4)4.2(b) (4)Control Item


4.3

DPBS

Test SystemMus musculus: 12 female BALB/c mice at an age of 9 weeks at study start witha body weight of approximately 25 g

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Materials

Table 3: Materials

Product nameApplication/specification

Article no.

Working
dilution

ProviderDulbecco’s
phosphate-bufferedsaline (DPBS)

Buffer control

14190-094

1×Thermo Fisher
Scientific4144150

N/A

BD

324824

N/A

BD

122799-10

150 mg/kg

Perkin-Elmer

Blood sampling2021-0131

N/A

Sarstedt

In vivo BLI imager

-

N/A

ELISA

439454

N/A

Positive control

E1701

100 ng/µL

Promega

10×
Chromogenic substrate forhorseradish peroxidase

B6429

1×

Sigma-Aldrich

4380H

N/A

BIOTREND

S8761100 ml

N/A

Sigma-Aldrich2104-50 ml

N/A

Sigma-Aldrich

61870
33214APT-2

N/A
N/A

Gibco
BiotekN/A

Mabtech

ELISpot plate reader-

N/A

Cell culture medium

61870

N/A

PROCARTAPLEX 10
PLEX

PPX-10 MXU63C9

N/A

Bio-Plex 200
Anti-firefly luciferase
antibody (mAb21),

Multiplex reader-

N/A

CellularTechnology Ltd.
Gibco
Life
Technologies
GmbH
Bio-RadAssay control ELISA

ab64564

1:1,000

Abcam

Mouse IgG isotype

Assay control ELISA

0107-08

Goat anti-mouse IgG
HRP

Secondary antibody ELISA

115-035071

Syringes 0.3 mL 30 G
Syringes 0.5 mL 29 G
Luciferin
Microvette 500 Z-gel
tubes
Xenogen IVIS®
Spectrum

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QuantiLum
recombinantluciferase
Casein blocking buffer
TMB ONE ECO-TEK
Sodium bicarbonate
HCl
RPMI1640 medium
Biotek Epoch
Mouse IFN-ELISpotPLUS kit
ImmunoSpot® S5
Versa Analyzer
RPMI1640 medium
Multiplex

Insulin syringes for i.m.
application
Insulin syringes for i.p.
application
Substrate for in vivo
luciferase imaging

7.5% NaHCO3
Hydrochloric acid solution
volumetric, 0.1 M HCl
Cell culture medium
ELISA plate reader
Kit for enumeration of cells
secreting mouse IFN-

Caliper Life
Sciences
Thermo Fisher
Scientific

1:100 asstarting
dilution
1:15,000 (ifstored in50%
glycerol
1:7,500)

Southern
BiotechJackson

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Table 4: Software

Product name
Living image
Prism
Excel
Bio-Plex Manager (6.1)
Gen5
ImmunoCapture V6.3

Application
In vivo BLI quantification
AnalysisAnimal monitoring, raw data ELISA
Bio-Plex reader
Absorbance reader
ELISpot analysis

ProviderPerkin-Elmer
GraphPad Software Inc.
Microsoft Corp.
Bio-Rad
BiotekCellular Technology Ltd.

Table 5: Peptide pool for stimulation of splenocytes

Peptide pool MHC-I
Name
Firefly luciferase-1
Firefly luciferase-2
Firefly luciferase-3

Sequence
GFQSMYTFVVPFHHGFGMVALPHRTAC

Consensus sequences from Limberis et al. 2009

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4.5

Methods

4.5.1

Animal Care

4.5.1.1

General Information

BALB/c mice were delivered at the age of at least six weeks. Delivered mice were used
for experiments after approximately one week of acclimatization. All experiments andprotocols were approved by the local authorities (local welfare committee), conductedaccording to the FELASA recommendations and in compliance with the Germananimal welfare act and Directive 2010/63/EU. Only animals with an unobjectionable
health status were selected for testing procedures.
All animals were registered upon arrival in the lab animal colony management system
PyRAT (Scionics Computer Innovation GmbH, Dresden, Germany) and tracked until
death. Each cage was labeled with a cage card indicating the mouse strain, gender,
date of birth, and number of animals per cage. At the start of an experiment additional
information was added such as the project and license number, the start of theexperiment and details on interventions. Where necessary for identification, animals
were arbitrarily numbered with earmarks.

4.5.1.2

Housing Condition and HusbandryMice were housed at BioNTech SE’s animal facility under barrier and SPF conditions(An der Goldgrube 12, 55131 Mainz) in individually ventilated cages (Sealsafe GM500
IVC Green Line, TECNIPLAST, Hohenpeißenberg, Germany; 500 cm²) with amaximum of five animals per cage. The temperature and relative humidity in the cages
and animal unit was kept at 20-24°C and 45-55%, respectively, and the air change(AC) rate in the cages at 75 AC/hour. The cages with dust-free bedding made of
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debarked chopped aspen wood (Abedd LAB & VET Service GmbH, Vienna, Austria,
product code: LTE E-001) and additional nesting material were changed weekly.
Autoclaved sniff M-Z food (sniff Spezialdiäten GmbH, Soest, Germany; product code:
V1124) and autoclaved water (tap water) were provided ad libitum and changed atleast once weekly. All materials were autoclaved prior to use.

4.5.2

Animal Monitoring

Routine animal monitoring wa carried out daily and included in pection for deadanimals and control of food and water supplies. Each animal’s health was closely
assessed at least once weekly. The general physical condition was assessed withregard to the following parameters:

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








4.5.3

Body weight change
Macroscopic assessment of activity level/ behavior
Macroscopic assessment of general discomfort: drop in body temperature
determined by touch and by visual inspection of ears and paws. Ears and paws
appear pink in a healthy mouse, white in a mouse with discomfort indicated by
reduced blood circulation
Macroscopic assessment of fur condition and appearance of eyes, inspectionof body cavities/ fluids
Macroscopic assessment of irregularities in breathing ability
Indication of pain
Macroscopic assessment for signs of automutilation and or fighting

Endpoint of Experiment / Termination Criteria

Animals were euthanized in accordance with §4 of the German animal welfare act andthe recommendation of GV-SOLAS by cervical dislocation or by exposure to carbon
dioxide. Additionally, termination criteria applied according to the specification within
the respective animal test approval as listed below. Body weight losses exceeding
20%, or a high severity level in any of the parameters found in Section 4.5.2 were on
their own sufficient reason for immediate euthanasia.

4.5.4

Injection of Test and Control Items

Animals were anesthetized by inhalation of 2.5% isoflurane in oxygen and the injection
site (hind leg) was shaved. Buffer or dissolved test item was applied i.m. into themusculus gastrocnemius at a volume of 20 µL. All mice received 1 µg in each leg. Afterinjection and a short recovery phase from anesthesia, the animals were observed forany signs of discomfort due to the injection procedure.

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Bioluminescence Measurements

The mice were monitored over a period of 9 days using in vivo imaging system (IVIS)
measurements. Briefly, the Xenogen IVIS® Spectrum device was used for in vivo
imaging according to the manufacturer’s instruction. Approximately 6 h after LNP
administration and 5 min prior to imaging, animals were injected for the first time
intraperitoneally (i.p.) with luciferin (150 mg/kg, dosing volume: 300 µL, 29 G needle).
Mice were anesthetized (2.5% isoflurane/O2) and placed in the imager, first with thedorsal side exposed and then with the ventral side exposed, and luciferase activity wasmeasured. Images were taken with exposure time and sensitivity set to 1 s, 10 s, and1 min and bin 2, bin 4, or bin 8, respectively. The dorsal and ventral images wereanalyzed by visual inspection after aligning of sensitivities of each picture and used forillustration of findings. The images were analyzed using Living Image in vivo imaging
3.0 software, where the regions to be quantified (radiance) were drawn manually andcalculated automatically (region of interest, ROI), to follow kinetics of the total fluxes(p/s) over time in a GraphPad file.

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4.5.6

Blood Sampling via the Vena Facialis

Blood was sampled via the vena facialis according to SOP-030-074. In short, without
prior anesthesia, mice were held tightly and using a lancet, the v. facialis was
punctured in a precise and short movement. Blood was collected into Microvette 500
Z-gel tubes, subsequently the restraining grip was loosened. Blood samples were
centrifuged at 10,000 ×g (room temperature) for 5 min and serum transferred to a prelabeled 1.5 mL reagent tube before storage at -20°C.

4.5.7

Luminex-based Multiplex Assay (ProcartaPlex Multiplex
Immunoassay)

The assay was performed according to manufacturer’s protocol. Briefly, magnetic
beads were added to the provided 96-well flat bottom plate and the beads were washed(wash buffer 1×) with the help of a hand-held magnetic plate washer. The antigen
standard was reconstituted in universal assay buffer (1×), pooled in one tube, thevolume adjusted to a final volume of 250 µL, serial diluted (4-fold serial dilution steps),and 50 µL was added to the designated wells. Serum samples were diluted 1:1 withthe universal assay buffer and 50 µL added to the wells. The standard was measured
in duplicates and the serum samples in triplicates. The plate was incubated on a plateshaker at 500 rpm) for 2 h covered with a black lid. After three wash steps, 25 µL of
the ready-to-use detection antibody was added, incubated for 30 min on the shaker
and washed three times. Streptavidin-PE (50 µL) were added and the plate incubatedfor 30 min on the shaker and washed three times. The beads were resuspended in
120 µL reading buffer, the plate was sealed, and data were acquired in the Bio-Plex
200 Luminex system.

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Luciferase-specific ELISA

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Luciferase-specific IgGs in serum samples obtained on study days 1 and 9 weredetected using ELISA. Recombinant luciferase (100 ng/100 µL) protein was utilized tocoat MaxiSorp plates at 4°C overnight. Upon washing and blocking using casein-based
blocking buffer, serum samples were screened for luciferase-specific antibodies by
incubation on plates for 1 h at 37°C. An anti-firefly luciferase antibody (mAb21) as well
as a mouse IgG isotype were included as assay controls. Subsequently, plates wereincubated with horseradish peroxidase (HRP)-labeled secondary anti-mouse IgG
antibody for another 45 min at 37°C before 3, 3’, 5, 5’-tetramethylbenzidine (TMB) ONE
substrate was applied. Colorimetric detection was monitored and optical density readat 450 nm calculated to a wavelength reference of 620 nm (ΔOD 450–620 nm).

4.5.9

ELISpot Analysis

4.5.9.1

T-cell epitope prediction

The respective peptides for stimulation of splenocytes (Table 5) were used as
published by Limberis et al. 2009, where the authors mapped the dominant and minor
T-cell epitopes in BALB/c mice (GFQSMYTFV and VPFHHGFGM, VALPHRTAC,
respectively) for monitoring cellular responses in vivo. No modifications have been
added to the published peptides before peptide synthesis by JPT technologies GmbH.

4.5.9.2

Sample Collection and Processing

Spleens were removed on day 9 after euthanizing the mice, and single-cellsuspensions were prepared (SOP-030-078). In brief, the removed organs werepressed through a 70 µm cell mesh using the plunger of a syringe to release the cells
from the organ into a tube. After washing with PBS the cell pellet was incubated witherythrocyte lysis buffer, washed in PBS, and passed again through a 70 µm cell mesh.
Resulting cells were resuspended in medium and counted.

4.5.9.3

IFN-γ ELISpot Assay

The IFN- ELISpot assay was used to measure IFN- release after in vitro stimulationof T cells as an indicator for the induction of antigen-specific T cells. ELISpot analysis
was performed using the Mabtech Mouse IFN- ELISpotPLUS kit. Isolated splenocytes
were seeded to pre-coated ELISpot plates at 5 × 105 cells/well in 200 µL medium andstimulated with antigen-specific peptide pools composed of single peptides and a final
concentration of 2 µg/mL per peptide, predicted as described in Section 4.5.9.1
overnight in a humidified incubator at 37°C. As peptide controls, splenocytes wereincubated with 6 µg/mL of an irrelevant AH1 peptide derived from the endogenous
retroviral gene product envelope glycoprotein 70 (gp70; AH1: amino acids 6 to 14).
Splenocytes were incubated with medium alone as a negative control or with 2 µg/mL
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the assay. Spots were visualized with a biotin-conjugated anti-IFN- antibody followed
by incubation with streptavidin-alkaline phosphatase (ALP) and 5-bromo-4-chloro-3indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) substrate. Spot numbers werecounted and analyzed using the ImmunoSpot® S5 Versa ELISpot Analyzer, theImmunoCaptureTM image acquisition software, and the ImmunoSpot® Analysis
software version 5. The quality control (QC) function of ImmunoSpot analysis software
was used to limit false positive spot counts. All tests were performed in triplicate andspot counts were summarized as median values for each triplicate.

4.5.10Statistical Analysis090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)

GraphPad Prism 8 software (La Jolla, USA) was used for statistical analysis and figure
generation. All groups were compared by a one-way ANOVA with Tukey’s multiple
comparison post-test on each measurement day (area under the curve forbioluminescence assay, ELISA, and ELISpot analysis).

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5

RESULTS

5.1

Bioluminescence Measurements

Page 19 of 36

The biodistribution of luciferase expressed by the LNP-formulated modRNA after i.m.
injection was assessed by bioluminescence measurements. Mice received a total doseof 2 µg (b) (4) LNP8, or (b) (4) -formulated modRNA, the control group received
20 µL DPBS only.
Mice were monitored over nine days and Luciferase signal was recorded andquantified (Figure 1).

A

Time
6h

24 h

48 h

72 h

6d

9d

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Buffer control(b) (4)

modRNA-Luciferase
LNP8

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C

Luciferace signal over time(b) (4)10 10

Total Flux [p/s]

10 8
10 7
10 6

10 9
10 8
10 7

h

h

d

d

48

72

6

9

10 5

h

10 4

24

10 6

h

10 5

6

(b) (4)

10 10

10 9

Total Flux [p/s]

Total AUC

Time after injection
Buffer control(b) (4)

modRNA-Luciferase LNP8(b) (4)090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)

Figure 1: Bioluminescence measurement using the LNP-formulated modRNA encoding for luciferase
BALB/c mice were injected i.m. in the right and left hind leg with each 1 µg of LNP-formulated modRNA encoding
luciferase or DPBS only. A) At different time points after injection, the luciferase expression in vivo was measured
by luciferin application. After 9 d, the reporter expression dropped to background levels. B) Quantification of
luciferase signal over time and C) as total area under the curve (AUC, ±SD). p/s: photons per second

All formulations resulted in a modRNA-typical expression over time (Figure 1). Highest
signal was detected at the first time points after immunization at the injection site andthe signal declined slowly over time until day 9 (Figure 1B). Luciferase expressed by
the modRNA formulated with Acuitas LNP8 drained to the liver as visualized by(b) (4)luciferase expression at 6 h in the liver region(b)(4)(b) (4)
Acuitasformulations (Figure 1A and Figure 2).
Group mean luciferase expression from RNA formulated with Acuitas (b) (4) LNP8(b) (4)in the muscle at 6 h was approximately 1 × 109 p/s,(b) (4)Hence, Acuitas-formulated
modRNA started at about 20-fold higher signal levels, stayed more than 20-fold higher(b) (4)(b) (4)
until 72 h (~7 × 107 p/s for
LNP8 vs) and(b) (4)
declined then to a low level(b) (4)on day 9 (~3–5 × 105 p/s).
Area under the curve calculation allowed comparing overall expression levels over thecourse of the experiment (Figure 1C). Statistical significance was assessed by oneway ANOVA with Tukey’s multiple comparison post-test comparing all groups with(b) (4)each other. Total luciferase expression from modRNA formulated with(b) (4)
while total luciferase expression from modRNA formulated(b) (4)
(b)
(4)
with
LNP8 was approximately
1.5 × 109 p/s, respectively.
(b) (4)The difference between the area under the curve for
LNP8-formulated(b)(4)
modRNA compared to buffer control
was

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Page 21 of 36(b) (4)statistically significant (p < 0.0001).
(b) (4)

5.2

Liver Expression(b) (4)LNP8090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)As mentioned in Section 5.1, luciferase expressed by the modRNA formulated with
Acuitas LNP8 drained to the liver as visualized by luciferase expression at 6 h in the(b) (4)liver region
Acuita (b) (4)(b) (4) LNP8
(Figure 2A). Here the luciferase signal of modRNA formulated with
was quantified for better comparison. Group mean luciferase expression from RNA(b) (4)formulated with Acuitas (b) (4) in the liver at 6 h was
while
luciferase expression of RNA formulated with LNP8 was at about 4.94 × 107 p/s.
(b) (4)(b) (4)Hence, luciferase expression fromAcuitas(b) (4)compared to LNP8 (Figure 2B). The liver luciferase expression from RNA
formulated with Acuitas LNP8 dropped to 2.4 × 106 p/s at 24 h, while the luciferase(b) (4)signal from RNA formulated with Acuitas
No liver signal
was detected at 48 h post immunization. Statistical analysis was not performed.
(b) (4)(b) (4)

Figure 2: Bioluminescence measurement in the liver using the LNP-formulated modRNA encoding for
luciferase
BALB/c mice were injected i.m. in the right and left hind leg with each 1 µg of LNP-formulated modRNA encoding
luciferase. A) At 6 h, 24 h, and 48 h after injection, the luciferase expression in vivo was measured by luciferin
application. B) Quantification of luciferase signal in the liver over time (mean ±SD). p/s: photons per second.

5.3

Luminex-based Multiplex Assay

Activation of the innate immune system was assessed in a Luminex-based multiplex
assay (Procarta immunoassays). Serum samples (day -1 (pre), 6 h, and day 9) were
tested for levels of the following chemokines and cytokines: MCP-1, MIP-1, TNF-,IFN-, IFN-, IL-2, IL-6, IL-10, IL-1, IP-10 (Figure 3). No cytokines/chemokines were(b) (4)detected in the pre-serum.
(b) (4)(b) (4)

Immunization with LNP8 induced slightly increased levels of MCP-1, IL(b) (4)6, and IP-10 at 6 h post immunization

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Page 22 of 36(b) (4)All chemokine/cytokine levels dropped tobackground levels at day 9. Statistical analysis was not performed for this assay.
MCP-1(b) (4)

8
Serum MIP-1 (pg/ml)

100

50

0

6
4
2
0

e
pr

6h

d9

e
pr

d9

6h

e
pr

6h

d9

e
pr

6h

d9

e
pr

6h

d9

e
pr

Serum IFN- (pg/ml)

4
2

6h

d9

6h

d9

e
pr

6h

d9

d9

e
pr

6h

d9

0.8
0.6
0.4
0.2

d9

e
pr

d9

6h

e
pr

6h

d9

e
pr

6h

d9

e
pr

6h

d9

e
pr

6h

d9

e
pr

IL-2

IFN-

(b) (4)(b) (4)

1.0
Serum IL-2 (pg/ml)

10

5

0.8
0.6
0.4
0.2
0.0

0
6h

d9

e
pr

6h

d9

e
pr

6h

d9

e
pr

6h

d9

IL-6

Serum IL-10 (pg/ml)

0
e
pr

6h

0.0

d9

IL1-

IP-10

(b) (4)(b) (4)100
Serum IP-10 (pg/ml)

1.0

6h

d9

d9

6h

6h

e
pr

d9

d9

6h

0.2

pr
e

e
pr

0.4

pr
e

d9

0.6

6h

50

0.8

d9

100

6h

(b) (4)

1.0

150

e
pr

6h

IL-10

(b) (4)

200

e
pr

d9

e
pr

pr
e

d9

6h

6h

pr
e

e
pr

6h

d9

d9

6h

d9

e
pr

pr
e

Serum IFN- (pg/ml)

e
pr

0.0
6h

15

Serum IL-6 (pg/ml)

d9

0.8
0.6
0.4
0.2
0.0

80
60
40
20

(b) (4)(b) (4)

d9

6h

pr
e

d9

6h

pr
e

d9

6h

pr
e

d9

6h

d9

Buffer control

pr
e

6h

pr
e

d9

6h

pr
e

0
6h

Serum TNF- (pg/ml)

6

e
pr

Serum IL1- (pg/ml)

6h(b) (4)1.0

0090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)

e
pr

IFN-

TNF-

(b) (4)

8

d9

6h

pr
e

Serum MCP-1 (pg/ml)

MIP-1

(b) (4)150

modRNA-Luciferase LNP8

Figure 3: Activation of the innate immune system by LNP-formulated modRNA encoding for luciferase
BALB/c mice were injected i.m. in the right and left hind leg with each 1 µg of LNP-formulated modRNA encoding
luciferase or DPBS only. Serum samples (day -1 (pre), 6 h, and 9 d) were assessed for presence of indicatedchemokines/cytokines in a Luminex-based multiplex immunoassay.

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Luciferase-specific ELISA

Luciferase-specific IgGs in serum samples obtained on study days -1 and 9 were
investigated by ELISA.
Before immunization, no luciferase-specific IgGs were detected (day -1, Figure 4).
Treatment with modRNA with all tested LNP formulations did not induce the formationof luciferase-specific IgGs on day 9 post immunization.

Day 9 (1:10)

090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)OD (450-620nm)

Day -1 (1:10)
3

3

2

2

1

1

0

0

Buffer control(b) (4)(b) (4)(b) (4)
modRNA-Luciferase LNP8

Figure 4: Luciferase-specific IgG ELISA on days -1 and 9
BALB/c mice were immunized with 1 μg/leg luciferase-encoding modRNA on day 0. Serum samples were collectedon days -1 and 9 and the total amount of antigen-specific immunoglobulin G (IgG) was measured via ELISA. The
serum 1:10 diluted. Individual ΔOD values are shown by dots; group mean values are indicated by horizontal bars(±SD).

5.5

IFN- ELISpot Assay

Mice were euthanized on day 9 and splenocytes were isolated to assess T-cell
responses by ELISpot analysis. Splenocytes were stimulated with luciferase-specificpeptide pools (Table 5) and IFN- secretion was detected. Statistical significance wasassessed by one-way ANOVA with Tukey’s multiple comparison post-test comparing
all groups with each other. Control measurements were performed using an irrelevant
peptide pool, medium only, or Concanavalin A.
Stimulation of splenocytes with MHC I-specific peptide pools induced IFN- responses
in T cells of animals immunized with all modRNA LNP candidates (Figure 5). Groupmean values of 53 spots per 5 × 105 cells were counted for animals injected with buffer
control after stimulation with MHC I-specific luciferase peptide pools. The high spot
count in can be attributed to reactivity of T cells of one mouse in group 1. Splenocytesof this mouse react also to the stimulation with AH1, the negative control. The groupmean values are also 44 spots be 5 × 105 cells for the AH1 control, thus the response
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of the control group to the luciferase-specific peptide pool can be consideredunspecific. Group mean spot counts after stimulation with MHC I-specific peptide pools(b) (4)were(b) (4)519 spots per 5 × 105 cells for the group treated with LNP8formulated modRNA. The reactivity of splenocytes of the treatment groups to thenegative control was very low (6-8 spots per 5 × 105 cells), thus activation of T-cellswith luciferase peptides is highly specific in the treatment groups.
(b) (4)(b) (4)The differences between the groups
treated
LNP8-formulated modRNAs compared to buffer control were(b) (4)statistically significant (p = 0.0051 for(b)(4)
modRNA-luciferase LNP8). The
and LNP8 groups displayed significantly higher(b) (4)MHC I-specific IFN- secretion(b) (4)p = 0.0163 for modRNA(b) (4)luciferase LNP8).
(b) (4)

AH1

Luciferase
1000

Spots per 5*105 cells

090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)(b) (4)(b) (4)(b) (4)1000

Medium and ConA controls
2000
1500

500

500

1000
500

0

0
Buffer control(b) (4)0(b) (4)

MediumConA

modRNA-Luciferase LNP8

Figure 5: ELISpot analysis using splenocytes on day 9
ELISpot assay was performed using splenocytes isolated on day 9 after prime immunization. Splenocytes were
stimulated with MHC I-specific luciferase peptide pools and IFN- secretion was measured to assess T-cell
responses. Individual spot counts are shown by dots; group mean values are indicated by horizontal bars (±SD).

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CONCLUSION

After injection of LNP-formulated modRNAs, an approximately 20-fold higher luciferase
expression at the injections site was observed in a bioluminescence assay for(b) (4)
the GMP-ready modRNA-Luciferase LNP8 (b) (4)(b) (4)The difference between the area(b) (4)
under the curve for
LNP8-formulated modRNA compared to buffer control(b) (4)(b) (4)was statistically significant.
(b) (4)A multiplex assay showed that the innate immune system was temporally activated by(b) (4)(b) (4)
LNP8-formulated modRNA.
(b) (4)090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)

Treatment with modRNA with all tested LNP formulations did not induce the formationof luciferase-specific IgGs on day 9 as measured by ELISA.
However, a strong antigen-specific IFN- T-cell response measured by ELISpot assay(b) (4)on day 9 for
LNP8-formulated modRNAs, with statistically significant(b) (4)differences between these test groups and the buffer control

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DOCUMENT HISTORY

Reasons for changes compared to previous version:
Minor editorial changes, such as the correction of typing errors, are not listed.
Sections

Version 01

1
3.1

-

6
4.1

Version 02
More detailed
information
about LNP8
added
Table added

Reason for change
Clarification that LNP8 contains ALC-0159 and
ALC-0315
Details of LNP lipid component formulations

Version 02

Version 03

Reason for change

4.1-

Test item
information
updated

Diameter of LNP8 modRNA was corrected to
71 nm

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Sections

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REFERENCES

Limberis MP, Bell CL, Wilson JM. Identification of the murine firefly luciferase-specific
CD8 T-cell epitopes. Gene Ther. 2009;16(3):441-7.
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 SelfAmplifying mRNA. Molecular therapy. Methods & clinical development. 2018; 12,32-46.
Pardi N, Hogan MJ, Pelc RS, Muramasu H, Andersen H, DeMaso CR et al. Zika virus
protection by a single low-dose nucleoside-modified mRNA vaccination. Nature. 2017;
543 (7644), 248-251.

090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)

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 atmuch lower doses. Molecular therapy: the journal of the American Society of Gene
Therapy. 2017; 26 (2), 446-455.

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APPENDIX

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Appendix 1: Animal Observations-5 days p.a.
09.01.2020
Lab IDweightin ghealth

1-1
1-2
1-3
2-1
modRNA2-2
Luciferase (b) (4)2-3
3-1Acuitas LNPs
modRNA
3-2
luciferase
3-3
4-1
modRNALuciferase LNP8
4-2
(GMP-ready)
4-3

21.4
22.5
23.0
20.7
21.5
21.2
21.3
21.2
20.9
22.2
21.6
20.5

No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations

treatment

Buffer control

1 days p.a.
15.01.2020
weight % change
in gweight
21.7
22.8
23.3
21.0
22.5
21.4
21.5
21.2
21.1
23.2
22.4
20.8

101.0
101.2
101.4
101.4
104.6
100.8
100.8
99.9
101.0
104.7
103.8
101.3

3 days p.a.
17.01.2020
healthNo observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations

weight % change
in gweight
21.5
22.2
23.5
20.9
22.3
21.3
21.4
21.0
22.3
22.9
22.5
20.9

100.4
98.6
102.3
100.7
103.8
100.4
100.5
99.1
107.0
103.2
104.3
101.7

6 days p.a.
20.01.2020
healthweightin g% changeweighthealthNo observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations

21.7
22.7
23.4
20.8
23.2
21.3
21.0
21.0
21.7
23.5
21.9
20.9

101.3
100.8
101.5
100.3
107.9
100.5
98.4
99.2
104.1
106.1
101.6
101.9

No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations
No observations

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Appendix 2: Certificates of Analysis090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)(b) (6)(b) (6)

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Appendix 3: Raw Data IFN- ELISpot
Plate 1: Left, luciferase peptides; right, AH1 ELISpot (irrelevant peptide)(b) (4)090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)(b) (4)(b) (4)(b) (4)(b) (4)
Group 1: Buffer control; Group 2:(2x1 µg); Group 3:(2x1 µg); Group 4: modRNA-luciferase LNP8 (GMP-ready) (2x1 µg)(b) (4)

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Appendix 4: Statistical Analysis
Bioluminescence assay
Group mean values, bioluminescence assay, luciferase signal over time(b) (4)(b) (4)

Time point

Buffer control

modRNALuciferase LNP8

6h
24 h
48 h
72 h
6d
9d

N=3
128046,667
227766,667
139995132585
162383,333
76573,333

N=3
1,2589e+009
7,310667e+008
2,1038333e+008
7,8667e+007
2920333,333
509000Please note that commas are used as decimal separators.

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Descriptive statistics, bioluminescence assay, area under the curve(b) (4)

Number of values
Minimum
Maximum
Range
Mean
SD
SEM

(b) (4)

Buffer control

modRNALuciferase LNP8365779088990823211876504011705867583

31352000000
1848000000
496000000
1652666667
264244836
152561827

Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of themean.

One-way ANOVA with Tukey’s multiple comparisons post-test, bioluminescenceassay, area under the curve
ANOVA summary
F
P value
P value summary
Significant diff. among means (P < 0.05)?
R square80,68<0,0001****
Yes
0,9680Strictly Confidential

FDA-CBER-2021-5683-0013957

R&D Report R-20-0072

Tukey’s multiple
comparisons test

Version 03

Mean diff.

95,00% CI of diff.

Page 34 of 36

Significant? Summary

Adjusted P
value

Yes

<0,0001(b) (4)(b) (4)

Buffer control vs.
modRNA-Luciferase -1651901626
LNP8-2072407467 to 1231395786****(b) (4)(b) (4)090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)(b) (4)

Please note that commas are used as decimal separators. F: F-statistic. P values ≤ 0.05 indicate statisticallysignificant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.

Luciferase-Specific ELISA
Descriptive statistics, luciferase-specific ELISA, day 9(b) (4)

Number of values
Minimum
Maximum
Range
Mean
SD
SEM

(b) (4)

Buffer control

modRNALuciferase LNP8

30,01100,01900,00800
0,01530,00404
0,00233

30,01000,02200,01200,0177
0,00666
0,00384

Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of themean.

Strictly Confidential

FDA-CBER-2021-5683-0013958

R&D Report R-20-0072

Version 03

Page 35 of 36

One-way ANOVA with Tukey’s multiple comparisons post-test, bioluminescenceassay, area under the curve
ANOVA summary
F
P value
P value summary
Significant diff. among means (P < 0.05)?
R square0,45970,6520nsNo0,1209No post-test for non-significant main test.

ELISpot analysis
Descriptive statistics, ELISpot analysis, day 9

090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)(b) (4)

Number of values
Minimum
Maximum
Range
Mean
SD
SEM

(b) (4)

Buffer control

modRNALuciferase LNP8

3
4,00
148
144
53,0
82,3
47,5

3
381
749
368
519
201
116

Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of themean.

One-way ANOVA with Tukey’s multiple comparisons post-test, bioluminescenceassay, area under the curve
ANOVA summary
F
P value
P value summary
Significant diff. among means (P < 0.05)?
R square19,90
0,0005***
Yes
0,8819Strictly Confidential

FDA-CBER-2021-5683-0013959

R&D Report R-20-0072

Tukey’s multiple
comparisons test

Version 03

Mean diff.

95,00% CI of diff.

Page 36 of 36

Significant? Summary

Adjusted P
value

Yes

0,0051(b) (4)(b) (4)

Buffer control vs.
modRNA-Luciferase -465,7LNP8-769,0 to -162,3**(b) (4)(b) (4)090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)(b) (4)

Please note that commas are used as decimal separators. F: F-statistic. P values ≤ 0.05 indicate statisticallysignificant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.

Strictly Confidential

FDA-CBER-2021-5683-0013960

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Anlagen:Freitag, 27. November 2020 07:24(b) (6)(b) (6)signatures: R-20-0072v3.0 (BNT162)
R-20-0072 Report V3.0 Bioluminescence in vivo_final_sig.pdf

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Hello (b) (6)With this email I’m giving my approval for the R&D report R-20-0072 version 3 update.
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BioNTech SE

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