Dyadic International : ECI Microbial Engineering January 2021

C 1 T E C H N O L O G Y P L A T F O R M

C 1 - A L O W - C O S T P R O D U C T I O N

P L AT F O R M T O C O M B AT

T H E C O V I D - 1 9 PA N D E M I C

R o n e n T c h e l e t, M a r k E m a l f a r b , M a t t h e w J o n e s

E C I C O N F E R E N C E O N M I C R O B I A L

E N G I N E E R I N G

M i c r o b i a l - b a s e d T o o l s t o c o m b a t C O V I D 1 9

January 26, 2021

Next Generation Protein Expression Biotech

Proprietary & Patented C1 gene expression platform technology

Competitive advantages

Validating partnerships

Opportunistic business development

Experienced management

Designed to bring biologic vaccines and drugs to market faster, in greater quantities, at lower cost

Robust and rapidly expanding scientific data that demonstrates high productivity stability and purity for a growing number of protein classes

Well-established, global biological R&D organizations, top-tier animal and human health pharmaceutical companies, as well as governmental agencies

Emphasis on large and growing addressable human and animal health markets, many shots on goal including vaccines and antibodies for infectious diseases and therapeutic proteins for diabetes, oncology and arthritis

Highly experienced and energized management team and board of directors driving process and execution excellence

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C1 GENE EXPRESSION PLATFORM

COMPETITIVE ADVANTAGES

C1 Competitive Advantages

Robust Gene Expression Platform Offers A Number Of Competitive Advantages Over Existing Technologies

Purity

Productivity

Robustness

Speed

Cost

High retention of target secreted protein through downstream processing

No requirement for viral inactivation

Robust & versatile growth conditions

High yields of secreted protein

Low viscosity [unique morphology]

At scales ranging from laboratory microtiter plates, shake flasks, single use and/or stainless- steel bioreactors.

Develop stable g/l C1 cell lines in ~7 weeks Production savings of ~30 days over CHO

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

High yields and fast production times can reduce cost and shrink manufacturing footprint Requires only low-cost cGMP synthetic media

No requirement for viral inactivation, simplifies processing compared to CHO, saving time, money

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C1 Expression Technology

		C1 Site Directed Transformation Method leads to quick generation of stable C1 cell lines
Transformation
	• Set of strong promiscuous promoters native and synthetic are available
efficiency
		• HC and LC can both be integrated into the same site
		• Stable single-copy integration
		• No need for transient stage
		• No need for induction

C1 Genome

HPH (3')

• Transformation procedure based on chemical (PEG) method with protoplasts or electroporation
• Frequencies for 1μg DNA: ~ 20 colonies
• • Not more than 20 transformants for site specific integration have to be screened in order to identify the right clones (usually >50% of the transformats have the expected productivity and quality

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C1 Expression Technology

Fed-batch

Process

• fully defined cheap medium
• fed-batchtechnology 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
• 1L to 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

From MTP to Large scale mAbs productivity

24 wells MTP - 1mg/4ml

1L fermentor - 1.7/g/l/d

30L fermentor - 2.4 g/l/d

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C1 GENE EXPRESSION PLATFORM

rVaccines Production Platform

Leveraging Third Party Funding to Advance C1 Platform

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 data and guidance on how to improve C1 for mass antigen production.
• Through collaboration with ZAPI scientists and others, C1 has a potentially important role in helping to combat pandemics.
• SBV antigen produced from C1 was more stable and produced at ~300 times greater levels than the SBV antigen produced from baculovirus.
• C1 expressed SBV antigen was very effective (Full Protection). ZAPI Animal studies concluded that Dyadic's C1 expressed SBV antigen demonstrated very strong performance in protecting both cattle and mice from the Schmallenberg virus.
• ZAPI is expected to fund additional animal trials in 2021 with C1 expressed antigens (SBV and RVFV)
• ZAPI success led to fully funded collaborations with several leading global animal & human health companies and governmental agencies

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-trackregistration of developed products to combat epidemic zoonotic diseases that have the potential to affect the human population.

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Success in Expressing High Level of SBV Antigen for ZAPI

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 and protocols to fast-track registration of developed products to combat epidemic Zoonotic diseases that have the potential to effect the human population.

• SBV causes congenital malformations and stillbirths in cattle, sheep, goats, and alpaca.
• An antigen against Schmallenberg Virus (SBV) that was developed by ZAPI group, was expressed by C1.
• Production level reached 1.8 g/L in 7 days fermentation - 300 fold higher than in Baculovirus.

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Success in mice and cattle challenge tests

Mice	Challenge D35	cattle
trials		trials

Challenge D35

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.

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

Doses per batch (20ug/dose)	100 K	1 000 K	10 000 K
Total volume (g)
2	20	200
Productivity (g/L)	1,75	1,75	1,75
Recovery (%)	75	75	75
Working volume (%)	80	80	80
Fermentation volume needed	2L	20L	200L
for 1 batch run with C1

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Successful Execution of Government Sponsored Collaborations

Positions Dyadic to Enter Clinical Trials

Israel Institute for Biological Research (IIBR)

• Entered into initial collaboration January 2018
• Focused on advancing C1 expression platform for the development and manufacture of recombinant vaccines and neutralizing agents comprising targeted antigens and monoclonal antibodies, to combat emerging diseases and threats
• A proprietary IIBR Fc-fusion enzyme has been expressed using C1 technology provides certain countermeasures against nerve agents such as sarin and VX gas
• February 25, 2020 expanded collaboration with the IIBR to combat emerging diseases including collaborating on a potential rVaccine candidate to combat COVID-19 outbreak

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DEVELOPING NEW PLATFORM

C1 Technology to support the global combat against Covid 19

Coronavirus Spike RBD Is A Key Target For Potent Neutralizing Abs

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

C1 fermentation is based on Fed-batch technology with glucose feeding and cGMP synthetic media Fermentation was run for 5 days in the Ambr250 system and in 5L fermentor scale

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

Ongoing fermentation process optimization led to even greater productivity ~ 3 g/l in 4 - 5 days

Receptor binding domain:

Single folded polypeptide chain

All potent neutralizing Ab target the RBD

Ag minimization -> focused immune response

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Evaluation Of RBD Produced By C1

Biomolecular Binding Kinetics Assays:The equilibrium

01 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.

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

02 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.

Recently - mice study confirmed that RBD antigen produced

03 by C1 induced the production of neutralizing antibodies against the SARS-CoV-2 in mice. Additional animal studies are underway to assess the immunogenicity and protective efficacy upon challenge in hamsters and transgenic mice expressing the Human Ace2 will be infected with the SARS- CoV-2 virus.

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

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Mice study with C1 expressed SARS-CoV-2 RBD vaccine candidate - Results

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

				ELISA
	6	1st	2nd	2nd	3rd
dilution	5
4
3
serum
2
1
log10
0
	-1
	19	dpv	28	dpv	43	dpv	50	dpv
	Day after first vaccination

Direct RBD ELISA.

Serum 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.

1st	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

PRNT

	6	128,000
	32,000
		22,627
NT50	5	11,310
8,000
log10	4	8,000
4,000
		4,000
		4,000
		2,828
	3	50 dpv

PRNT

Serum 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.

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Commercial Scale Production of RBD Antigen by C1

Predicted RBD fermentation capacities for different dose requirements without and with coupling to multimeric protein scaffold particle (MPSP) (Calculated according to RBD 3.0 g/L estimated RBD yield at commercial scale)

We believe that the C1 expressed RBD of the SARS-CoV-2 Spike Protein has the potential to be an effective low-cost vaccine candidate that can be rapidly manufactured at flexible commercial scales to help combat the COVID-19 pandemic.

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Fast & Stable High Producing C1 Cell Line Development

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

Rapid Response

High Doses

Scalability

Affordability

Original RBD version

The same rapid

New RBD version

• Set of strong promoters native and

method will be

synthetic

needed to

• No need for induction

construct new

cell line with the

• Stable single-copy integration

new gene

• No need for transient stage

C1 genome

C1 genome

1 week

3 weeks

1 week

2 weeks

•

Rapid Development Timelines

Strain

MTP ferm., DSP

1-30 l scale

•

High Productivity - large quantities

Gene synthesis

Plasmid

construction and

and analytics

fermentation

construction

re-isolation for

& RCB

from RCB, DSP

•

Purity

monoclonality

generation

and analytics

•

Stability

•

Robust Manufacturing Process

Sending samples

cGMP grade strain

and process

•

Flexible Commercial Scales

for evaluation,

animal studies

characterization,

•

Low Cost

MCB,

If the COVID-19 virus mutates and a second-generation vaccine/mAb is needed, C1 can be
used to rapidly develop and manufacture it in larger quantities more affordably.	18

SARS-CoV-2 Proteins Rapidly & Stably Expressed by C1

Different SARS-CoV-2 potential rVaccines were successfully expressed by C1:

• SARS-CoV-2RBD
• SARS-CoV-2RBD Spy Tag (to generate nanoparticles)
• SARS-CoV-2Spike Protein
• SARS-CoV-2Spike Protein with Spy Tag (to generate nanoparticles)
• SARS-CoV-2RBD FC

• SARS-CoV-2 mAb

Neutralizing mAb

Nucleocapsid protein (NP)

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C1 SARS-Cov-2 Vaccine & mAb Programs Ongoing

Nine programs globally

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Acknowledge

Dyadic Inc.

Mark Emalfarb,

Matthew Jones,

Ping Rawson,

Julie Latiuk,

Yuka Simmons,

Heidi Zosiak,

Sue Bristow

VTT Finland

Markku Saloheimo,

Anne Huuskonen,

Chris Landowski,

Marika Vitikainen,

Georg Schmidt,

Anssi Rantasalo,

Veera Korja,

Marilyn Wiebe,

Hanna Kuusinen,

Karita Viita-aho

Kaisa Roine

DYADIC INFORMATION

R&D: Helsinki

BD&L: LondonR&DManagement: Budapest

R&D: Valladolid

HQ: Jupiter, FL

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C1 Technology Can Be Applied To A Broad Set Of Therapeutics

Versatile "Workhorse" Capable of Manufacturing Proteins Quickly and at Low Cost

Impressive Yield and Purity Demonstrated for Therapeutic Proteins1

Fc-Fusion

mAbs

Fab (Certolizumab)

Trispecific

15.3 g/l 1

24.5 g/l 1

14.5 g/l 1

168 Hours

168 Hours

164 Hours

6.12 g/l1

2.58 g/l/day

3.1 g/l/day

2.1 g/l/day

144 Hours

1.02 g/l/day

Hyper Productivity for Antigen Classes Routinely Used in Vaccines

Hemagglutinin (HA)

Antigen

Virus-Like Particles

413 mg/l 1

3,500 mg/l1

2,200 mg/l1

137 Hours

96 Hours

110 Hours

72 mg/l/day

875 mg/l/day

500 mg/l/day

1. Data is from non glycoengineered C1 Strains using different protease minus C1 strains	22