C1-CELLS: A TRANSFORMATIONAL PLATFORM

THAT TRANSCENDS THE LIMITS OF LEGACY

PROTEIN PRODUCTION TECHNOLOGIES

Dyadic Thermophilic Filamentous Fungus (C1 platform)

Recombinant Production of Glycoprotein Antigen Vaccines, Antibodies &

Other Therapeutic Protein Products

BIO International Convention Digital Conference

June 10-11 & 14-18, 2021

1

C1 Protein Production

Platform

Biopharma industry-leading global commercial,

government & academic strategic collaborations

Building a more efficient biomanufacturing platform to

serve a global population more affordably

Opportunistic participation in product development of

vaccines, biotherapeutics for infectious, oncology, &

autoimmune diseases

Novel synthetic biology tools for developing diagnostic,

prophylactic, and therapeutic products

C1 is ideally suited to help meet the global demand for Health

Equity including multivalent vaccines & multi antibody cocktails

GLOBAL HEALTH ACCESS, AFFORDABILITY & EQUITY

3

PuritiesHigh retention of target secreted protein(s) or other C1-cell bioproduct(s) through

downstream processing

No viral or endotoxins

ProductivityRobust & versatile C1-cell growth conditions

High yields of C1-cell secreted protein and other products

Low viscosity [unique morphologies]

SpeedsDevelop stable C1-cell lines in ~7 weeks producing recombinant proteins at grams/liter

C1-cell production savings of ~30 days over CHO-cell production costs with very expensive media

Manufacturing ~ 3-4 batches of mAbs at the same time it takes to make 1 batch using CHO-cells

Robustness Scales ranging from laboratory microtiter plates, shaker flasks, single use and/or

stainless-steel bioreactors

Robust Recombinant Protein Production Platform Offers Competitive Advantages Over Existing Techs

C1-Cell Recombinant Protein Products With Competitive Advantages

Costs

4

High yields and rapid manufacturing cycle times reduce C1 production costs and significantly

reduce manufacturing footprint

C1 Protein Production Platform Overview

• The C1 platform offers several advantages to address therapeutic and vaccine production :

• Highly productive and stable cell lines that are suitable for the rapid development and production of antigens without the need for transient stage

• Highly efficient, robust & versatile low-cost fermentation processes

• Rapid fed-batch fermentation cycles with 12 – 14 days from seed flask to finished fermentation

• Secreted proteins with high proportions of human glycoforms

C1 is characterized by a morphology

change to a fragmented non

sporulating mycelial phenotype.

This morphology results in high

titer/low viscosity fermentations

Systematic deletion of protease genes based on:

▪ Isolation and identification of extracellular proteases

▪ C1 protease library in Pichia pastoris

▪ Effect of different protease inhibitors on protease

activity

▪ mRNA sequencing data

▪ Protease gene annotation

Increasing

Stability

5

Current status• G0 strains – best G0 levels 84% on Nivolumab in fermentation

(98% in SF)

• G2/G1 strains – best strain has 26% G2, 45% G1 and 27% G0 on

Nivolumab in fermentation

• Fucosylation – best strain has 29% G0F, 61% Man3GlcNAcFuc and

5% G0 on Nivolumab in fermentation – up to 95% Fucosylation

efficiency

• G1F/G2F strains are under construction

Glycan

MT330, +38 C

M3848, Niv G0F (C1 Kre2a ss)

Day 7 (166 h)

Response Amount (%)

M3 11313958 4,77

M3-GlcNAc 376751 0,16

M3-GlcNAc-F 144330411 60,86

G0 11335452 4,78

G0F 69138334 29,16

?

sum:

637308 0,27

237132214 100

C1 typical Glycan structure New C1 Glycan Structure

G0 G0F G2 G2F

C1 Protein Production Platform – N-glycoengineering

6

Fast & Stable High Producing C1 Cell Line Development

C1 Site Directed Transformation Method leads to quick generation of stable C1 cell lines

• Set of strong promoters native and

synthetic

• No need for induction

• Stable single-copy integration

• No need for transient stage

Rapidly develop COVID-19 Variant Antigen Multivalent Vaccine Candidates which can be manufactured in microbial single use or stainless-steel bioreactors in larger quantities more affordably.

Rapid Response

High Doses

Scalability

Affordability

• Rapid Development Timelines

• High Productivity – large quantities

• Purity

• Stability

• Robust Manufacturing Process

• Flexible Commercial Scales

• Low Cost

Original RBD version New RBD versionThe same rapid method will be

needed to construct new

cell line with the new gene

1 week 3 weeks 1 week 2 weeks

Plasmid

construction

Strain

construction and

re-isolation for

monoclonality

MTP ferm.,DSP

and analytics

& RCB

generation

1-30 l scale

fermentation

from RCB, DSP

and analytics

Gene synthesis

Sending samples

for evaluation,

animal studies

cGMP grade strain

and process

characterization,

MCB,

C1 genome C1 genome

7

C1 Biomanufacturing – Single Use / Stainless Steel Bioreactors

Fed-batch

Process

From MTP to Large scale

mAbs productivity

Seed Tank

200L – 500m3

FERMENTER

Harvest Tank

Seed flask

Inoculum

Defined

medium

NH4OH for

pH control

Glucose

feeding

Processing

& RecoveryPackaging Assembly Filling Formulation

centrifugation,

Protein A,

Purification

Fed-Batch

technology

48 hrs 24 hrs

▪ fully defined cheap medium

▪ fed-batch technology with glucose feeding

▪ wide range of conditions available pH: 5-8, Temp: 20 - 45°C

▪ low viscosity culture due to unique morphology in the fermenter

▪ (typically) 4-7 day process

▪ 1Lto 500,000L fermentation scale, stainless steel or single use stirred tank fermenters

▪ At the end 30-40% biomass, 60-70 % supernatant (titers refer to the supernatant)

▪ protein production requires no inducer

▪ protein is (typically) secreted to the media

24 wells MTP – 1mg/4ml

1L fermentor – 1.7/g/l/d

30L fermentor – 2.4 g/l/d

120-168 hrs

8

Case Studies – Relevant Protein Titers Achieved in C1

Fc-Fusion15.3 g/l

168 Hours2.58 g/l/day

Influenza HA up to 413 mg/l

In 137 h(72 mg/l/day)

trispecific up to 6.12 g/l

144 Hoursup to 1.02 g/l/day

Virus-like Particles (Secreted)

up to 2400 mg/L In 110 h

(500 mg/l/day)

Therapeutic Proteins

Virus antigen2-3 g/LIn 96 h

(500-750 mg/l/day)

Vaccines

(Note: Results are from non glycoengineered C1-cell lines using different protease strains, HA data from 2015)

mAbsup to 24.5 g/l

168 Hoursup to 3.5 g/l/day

Fab (Certolizumab)up to 14.5 g/l

164 Hoursup to 2.1 g/l/day

2015

9

C1 GENE EXPRESSION

PLATFORM

rVaccines Production

Platform

Animal Health

10

Animal Health: ZAPI Chose C1 Platform in Competitive Process

• Animal health is a rapidly growing market for both companion and farm animals.

• Vaccine & Drug development in animal health typically has a shorter regulatory timeframe and the cost of the vaccines and drugs is important.

• Dyadic has been involved in the Zoonoses Anticipation Preparedness Initiative (ZAPI) since 2015.

• ZAPI collaboration has helped generate PoC safety, efficacy and challenge data and demonstrated very high antigen production from C1-cells.

Zoonotic Anticipation Preparedness Initiative (ZAPI), is a research and development program sponsored

by the EU . The goal is to developing a platform suitable for the rapid development and production of

vaccines and protocols to fast-track registration of developed products to combat epidemic zoonotic diseases

that have the potential to affect the human population.

Leveraging Third Party Funding to Advance C1 Platform

ZAPI Final Cell Line Decision

C1 was chosen as ZAPI production host for antigens that fulfils ZAPI

goals:

(i)to be fast in producing sufficient quantities of doses when the idea is to deliver high number of doses under 4-6 months and

(ii)to be able to produce sufficient quantities of doses at a very low cost using reduced fermentation capacity.

Potential advantage of commercial scale production of SBV antigen by C1

The production volume that will be needed to produced 1 batch of 100K, 1,000K and 10,000K SBV doses with C1 (1.75 g/L)

11

Success in Expressing High Level of SBV & RVFVAntigens

• ZAPI, is a research and development program sponsored by the EU with the goal of developing a platform suitable for the rapid

development and production of vaccines to fast-track registration of developed products to combat epidemic Zoonotic diseases.

• SBV (Schmallenberg Virus) causes congenital malformations and stillbirths in animals. An antigen against SBV was expressed by

C1. Production level reached 1.8 g/L in 7 days fermentation – 300 fold higher than in Baculovirus

• RVF (Rift Valley Fever ) is a viral disease of humans and livestock that can cause mild to severe symptoms. Animals such as cows, sheep, goats, and camels may be affected. An antigen against Rift Valley Fever Virus (RVFV) was expressed by C1.

SBV yields: 1, 800 mg/L(time point 121h)

C1 Fermentation Baculovirus Fermentation

SBV yields: 6 mg/L(time point 192h)

100n

g

200n

g

300n

g

9Δ 8Δ GcN+C

72h

94h

0,28µl 0,38µl culture supernatant

250kD

150

100

75

50

37

25

20

15

10

76h

99h

12

1h

14

6h

X300

RVFV Gn_DVII-SpyCatcher-C-tag

SBV

RVFV yield: 1,24 g / L

RVFV

12

Coupling process

SBV GcN+C-SpyTag + SpyCatcher-AldolaseCoupling overnight needed to reach best resultsCoupling of inactivated, concentrated material (BEVS expressed) SBV detection and quantification in JESS

E2-SpyTag +SpyCatcher-RVFVCoupling kinetics look similar

13

Secretion of subunits in C1 is a key advantage for yields and DSP. C1 fungus is the chosen ZAPI production host for the subunits. It is:• Fast in producing high amounts of doses under 4-6 months

• Capable to produce sufficient amount of doses at a very low cost and reduced fermentation volume capacity

Drivers for the coupling process step:

• Fast coupling

• Single step process

• Ensure coupling of all subunits on MPSP

• Flexible coupling ratio if needed

• “structured” active ingredient

Coupling

C1 Expressed SBV Antigen - Success in Mice and Cattle Challenge

The C1 expressed SBV antigen that was assembled to Nano-particle expression molecules was tested in animal tests:• All immunized mice and cattle survived challenge infection without any clinical signs of disease.

• Protection was conferred even after only one immunization.

14

Mice trials

Cattle trials

Challenge D35 Challenge D35

0

20

60

80

100

0 1 2 3 4 5 6 7 8 9 10

days postinfection

%su

rviv

al

D)control

mock

GcHS 2x + A

GcHS 1x + A

LS-GcHS 1x + A

40 LS-GcHS 2x + A

Survival

4

2

0

10

8

6

0 1 2 3 4 5 6 7 8 9 10

days postinfection

[lo

g10]R

NA

co

py

nu

mb

ers

/ml

mock

LS-GcH

LS-GcHS

LS-Pept2

Viremia serum

Info about ZAPI Stakeholders Final Conference are available from the IABS website, and recording are available on YouTube:

h t t p s : / / z a p i - s t a k e h o l d e r s - f i n a l - c o n f e r e n c e . i a b s . o r g / c o n f e r e n c e - i n f o r m a t i o n . p h p ? p a r a g = s l i d e s

• D a y 1 – M o r n i n g , F e b r u a r y 4 , 2 0 2 1

• D a y 2 – M o r n i n g , F e b r u a r y 5 , 2 0 2 1

D a y 1 – A f t e r n o o n , F e b r u a r y 4 , 2 0 2 1 D a y 2 – A f t e r n o o n , F e b r u a r y 5 , 2 0 2 1

European Union Zoonosis Anticipation Preparedness Initiative

ZAPI Stakeholders Final Conference

• Many infectious diseases, including influenza and

Ebola, can be transmitted to humans from animals

(and vice-versa). Known as zoonoses, these

diseases represent a serious threat to both human

and animal health.

• ZAPI brings together experts in human and animal

health to create new platforms and technologies that

will facilitate a fast, coordinated, and practical

response to new infectious diseases as soon as

they emerge.

"Within the collaboration, we helped to validate a prolific

fungus, developed by one of our project partners, Dyadic, who

demonstrated that their C1 technology can be used to churn out

vaccines and antibodies in unprecedented amounts which has

the potential to further accelerate biologic manufacturing

processes," said project coordinator Dr. Jean-Christophe

Audonnet. Dr. Audonnet continued, "Dyadic and its C1 cell

line far exceeded our initial expectations at the start of the

program, turning in record antigen productivity for both

the SBV and RVFV antigens."

Dyadic’s C1 Gene Expression Platform Played a Key Role In The Success Of The Five-Year EU € 20 Million ZAPI Program

15

C1 GENE EXPRESSION

PLATFORM

rVaccines Production

Platform

Infectious Disease

16

SARS-CoV-2 Spike RBD Is A Key Target For Potent Neutralizing mAbs

• In ~2 months, we developed a C1 cell line expressing the Receptor Binding Domain (23kDa) of SARS-CoV-2 spike protein

• C1 stable cell line was developed that expressed the RBD originally at a level of ~ 1 g/L– no need for transient stage

• Fermentation optimization 2-3 g/l in 5 days in 22L fermenter

• C1 fermentation is based on Fed-batch technology with glucose feeding and cGMP synthetic media

• The RBD antigen was secreted to the media – no need for induction

• Transgenic mice challenge test demonstrated full protection

Advancing Towards Phase 1 clinical study Q3-Q4 2021

Receptor binding domain:

• Single folded polypeptide chain

• All potent neutralizing Ab target

the RBD

• Ag minimization -> focused

immune response

17

• SARS-CoV-2 proteins and neutralizing mAb successfully expressed by C1

• S-RBD

• S-RBD / Spy Tag (Nanoparticle)

• Full Spike / Spy Tag (Nanoparticle)

• Full Spike

• FC RBD

• MAb

• COVID-19 Variant candidates under development

• Infectious disease antigen targets expressed in C1 with preclinical PoC:

• Influenza HA

• MERS RBD

• SBV antigen (Schmallenberg)

• RVFV (Rift Valley Fever)

• IBVD-VLPs

Antigens and neutralizing mAbs for SARS-CoV-2 vaccines

Neutralizing mAbC1 / RBD Ctag C1 / RBD-SpyTag no CTag

HPLC analysis RBD SpyTag -

Purity 99.29%

Expression of secreted

Nanoparticles at several g/L

18

Stable C1-RBD-SpyTag Production Without C-Tag

HPLC analysis RBD SpyTag - Purity 99.29%

2,5

2

1,5

1

0,5

0

0,00 1,00 4,00 5,00

OD

45

0n

m

2,00 3,00

Concentration (Log ng/ul)

RBD-SpyTag

1Lscale fermentation

ELISA-ACE2 BindingAssay

• Stable C1 cell lines expressing RBD-SpyTag without C-Tag successfully developed• Production level in 1L fermentor reached 0.7-0.8 g/L in 55hrs• Efficient and simple purification steps with Spike Protein Resin was developed• Purity reached a level of 99.29% with minor losses• The RBD-SpyTag performed very well

1 - Mw marker 2 – EFT 55h 8µl3 – F.T. (15ml) 20µl4 – Wash I 30µl5– Elutions 1+2+3 in conc. 15µl 6 – Flow through 30µl7 – RBD std. (2µg total) 10µl

19

20

Evaluation Of RBD Produced By C1

OCTET assay: RBD antigen binding assay. A ACE2 receptor is immobilized on the

biosensor, followed by the binding of the RBD antigen. The binding coefficient is

measured by the Biosensor

In addition, all RBD neutralizing mAbs (that bind to different

RBD epitopes) that were identified in patients infected by

SARS-CoV-2 were efficiently bounded to C1 RBD-Ctag

antigen. This binding clearly demonstrates that C1-RBD

antigen was properly folded and has high potential to generate

immune response and protection against the SARS-CoV-2

virus.

Biomolecular Binding Kinetics Assays: The equilibrium

dissociation constant (KD) of C1 SARS-CoV-2-RBD-Ctag

binding to recombinant hACE2 was calculated to be 4.9

Nm, which is comparable to that of the CHO SARS-CoV-2-

RBD: 5.11 Nm.

Recently – mice study confirmed that RBD antigen produced

by C1 induced the production of neutralizing antibodies

against the SARS-CoV-2 in mice.

Immunogenicity and protective efficacy upon challenge in

transgenic mice expressing the Human Ace2 infected with the

SARS-CoV-2 virus were successfully completed

01

01

0103

02

01

Mice study demonstrated that the C1-RBD-CTag induced neutralizing antibodies at high level.

Direct RBD ELISASerum samples obtained at 19, 28, 43 and 50 dpv were tested in a direct ELISA assay. For each mouse (n = 10) serum dilutions scoring positive in the ELISA are plotted, bars represent geometric means for each sampling time-point.

PRNTSerum samples obtained at 50 dpv were tested in a PRNT against SARS-CoV2 on Vero E6 cells. NT50 values are plotted for each mouse (n=10) and the geometric mean value is indicated.

3

6

5

4

PRNT

50 dpv

log

10

NT

50

1st1st 2nd 3rd

Plaque reduction neutralization test (PRNT)

SARS-CoV-2 and Vero E6 cells

NT50 Dilution that neutralizes 50% of the virions

Conducted on pooled sera According to Titer

(GEOMEAN OF THE titers):

Range 1:1,600 -3200 (Low) - 1,280

Range 2: 6,400 -12,800 (Mid) - 5,120

Range 3: 25,600 - 51,200 (High) - 20,400

6

5

4

3

2

1

0

-1

ELISA

2nd 2nd 3rd

log

10

seru

md

ilu

tio

n

Day after first vaccination

128,00032,00022,62711,3108,0008,0004,0004,0004,0002,828

DYAI-100Vaccine

Candidate21

Predicted C1-RBD fermentation capacities for different dose requirements without and with coupling to multimeric protein scaffold particle (MPSP)

C1-expressed SARS-CoV-2 RBD has the potential to be an effective low-cost vaccine candidate that can be rapidly manufactured at flexible commercial scales

C1 productivity (3.0 g/L)Doses without MPSP (20µg and 20µg) Doses with MPSP (15µg+15µg)

10M 100M 1000M 10M 100M 1000M

Total volume (g) 400 4 000 40 000 400 4 000 40 000

Productivity (g/L) 3.0 3.0 3.0 3.0 3.0 3.0

RBD purification Recovery (%) 70 70 70 70 70 70

Coupling ration (MPSP:RBD) 3:1 3:1 3:1

Coupling recovery (%) 70 70 70

Total fermentation volume (%) 80 80 80 80 80 80

Calculated fermentation volume C1 (L) 238 2 381 23 810 85 850 8 503

22

SARS-CoV-2 (Infectious Disease) Strategy For Global Populations

Rapidly develop and manufacture affordable vaccines that are safe and effective that can protect against two or more of the current and future COVID-19 Variants as they emerge (i.e., tri-valent and tetra-valent vaccines).

Four critical Variant RBDs in preparation for C1-cell manufacturing of Dyadic-4TVATM Antigen-encoded SARS-CoV-2

vaccine candidate

Gene synthesis Plasmid construction

1 week

Strain construction and

re-isolation for

monoclonality

3 weeks

MTP ferm., DSP and

analytics & RCB

generation

1 week 2 weeks

1-30 l scale fermentation

from RCB, DSP and

analytics

Sendingsamples for

evaluation, animalstudies

cGMP grade strain and

process characterization,

MCB

MonoValent & MultiValent Recombinant Variant mAb SARS-CoV-2 Product Candidates

• We have executed an MTAwith top tier pharmaceutical company to perform a fully funded research & evaluation of a C1 produced SARS-

CoV-2 monoclonal antibody

• We anticipate entering into a collaboration with former ZAPI EU scientists and other parties to express one or more SARS-CoV-2

monoclonal antibodies with the objective leading to a Phase 1 Human Clinical Trial of a single mAb or a cocktail of more than one mAb

expressed from C1-cells.

23

Coordination of clinical trials for

anti-viral vaccines

Developed the C1-RBD

protein based rVaccine

Development of MonoValent

RBDcharacterization/evaluation,

Support with regulatory &

CMC consultation towards

accelerated licensure

Collaboration on preclinical

evaluation of the vaccine

candidates in small animal

cGMP manufacturing of

Toxicology & Clinical Trial

Material

Bio-Technology General (Israel)

a Ferring Company

Phase I with C1-cell SARS-CoV-2 RBD Recombinant Vaccine in 2021cGMP Drug Substance; Toxicology Ongoing; cGMP Drug Product (fill/finish); Initiating Anticipated Human

Phase 1 Clinical Trial of a C1 produced SARS-CoV-2 vaccine candidate - Prove Safety & Efficacy In Humans

24

C1 GENE EXPRESSION

PLATFORM

Influenza Antigens

25

Expression of Haemagglutinin (HA) in C1

Ability to Express Biologically active HA´s➢ 5 Recombinant HA´s have been expressed by C1:

Influenza Strain Expression Bioactive HA

New Caledonia, A (H1N1) yes yes

Texas, A (H1N1) yes yes

Puerto Rico, A (H1N1) yes yes

California, A (H1N1) yes yes

Florida, B yes yes

Agglutination Test:

Mice Study was conducted by Sanofi-Pasteur:

The full lengh rHA from A/New

Caledonia/20/99 (H1N1) strain

showed excellent immunogenicity

properties in mice without adjuvant

26

Seasonal Flu

University of Oslo consortium study to determine viability of C1 to express APC-targeted Influzena virus antigen proteins. C1 previously successfully tested in a Sanofi Pasteur influenza vaccine trial versus baculovirus.

2015 immunogenicity study of Recombinant Hemagglutinin (HA) from the A/H1N1/New Caledonia/20/99 strain with Sanofi Pasteur demonstrated that:

o (1) C1 produced r-HA was safe and well-tolerated in mice; and

o (2) the C1 produced r-HA was at least as immunogenic in mice as the baculovirus-rHA.

Plan to partner and advance development or seek government funding

27

mAb Production in the

C1 Platform

Biomanufacturing Capacity:

Newblockbuster mAbs could leave shortage

C1-Cells Disrupting Monoclonal Antibody Development & Manufacturing

• Three to four batches of C1 mAbs in same time as one batch of CHO mAbs

• Produce greater quantities from each batch more affordably

• Same binding and neutralizing properties

C1 Produced mAb’s are Stable and Correctly Folded

Purification by ProtA

C1-produced mAb

CHO-produced control

HC

LC

Input – precleared fermentation broth Fr

.4

Fr.5

Fr.6

Fr.7

Western blotting

400

ng

6

20

0n

gDays 2 3 4 5

Western Blotting

Surface Plasmon Resonance

➢ Typically stable mAb production levels are

between 5 – 10 g/L in 5-7 days

➢ Best productivity at Amber system:

o 24.5 g/L in 7 days (3.5 g/l/day)

➢ Best productivity so far at 30L scale:

o 20.6 g/L at 157 h (3.1 g/L/day)

Purification

CONFIDENTIAL 30

Comparison of Neutralizing Activity of mAb Produced in C1 & CHO

C1 mAb CHO mAb

Hemoglobin was measured at OD450 in supernatants

Hemolytic assay in rabbit RBC with alpha toxin of Staphylococcus aureus

The neutralizing activity assay demonstrated high similarity between

C1 and CHO produced mAb

31

32

C1 platform produces comparable therapeutic proteins as CHO while overcoming key production

limitations

C1 platform has potential to replace conventional mAb manufacturing

C1 produces stable and correctly

folded mAbs that have binding and

neutralizing properties to those

produced from CHO cells

Flexible production scale; C1 media <1/20 of the cost of CHO media

No viral inactivation required

Lower

Cost

C1 produces mAbs significantly faster (12-14 days) vs CHO cells (41-

54 days) / From frozen vial until the end of fermentation.

Faster

Production

C1 produces more product per batch and larger overall quantities

~ Potential to produce three to four batches using C1 in the same

timeframe as one batch using CHO cells

Higher

Yields

33

GLOBAL HEALTH ACCESS, AFFORDABILITY & EQUITY

Australia

India

Asia

Africa

(including Middle East)

North America

South America

Contact: memalfarb@dyadic.com