Document

SECURITIES AND EXCHANGE COMMISSION

Washington, D.C. 20549

Pursuant to Section 13 or 15(d) of the

Securities Exchange Act of 1934

Date of Report (date of earliest event reported): February 19, 2020

Dyadic International, Inc.

(Exact name of registrant as specified in its charter)

Delaware

(State or other jurisdiction of incorporation or organization)

000-55264

(Commission File Number)

45-0486747

(I.R.S. Employer Identification Number)

140 Intracoastal Pointe Drive, Suite 404
Jupiter, FL 33477

(Address of principal executive offices and zip code)

(561) 743-8333

(Registrant’s telephone number, including area code)

N/A

(Former Name or Former Address, if Changed Since Last Report)

Check the appropriate box below if the Form 8-K filing is intended to simultaneously satisfy the filing obligation of the registrant under any of the following provisions:


¨			Written communications pursuant to Rule 425 under the Securities Act (17 CFR 230.425)
¨			Soliciting material pursuant to Rule 14a-12 under the Exchange Act (17 CFR 240.14a-12)
¨			Pre-commencement communications pursuant to Rule 14d-2(b) under the Exchange Act (17 CFR 240.14d-2(b))
¨			Pre-commencement communications pursuant to Rule 13e-4(c) under the Exchange Act (17 CFR 240.13e-4(c))


Indicate by check mark whether the registrant is an emerging growth company as defined in Rule 405 of the Securities Act of 1933 (17 CFR §230.405) or Rule 12b-2 of the Securities Exchange Act of 1934 (17 CFR §240.12b-2).
Emerging growth company ☐
If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act. ☐

Securities registered pursuant to Section 12(b) of the Act


Title of each class	Trading Symbol(s)	Name of each exchange on which registered
Common Stock, par value $0.001 per share	DYAI	The NASDAQ Stock Market LLC

Item 8.01 Other Events

On February 21, 2020, Dyadic International, Inc. (the “Company”) announced that data presented at the 15th European Conference on Fungal Genetics (“ECFG15”) demonstrated its C1 strain has been glyco-engineered to achieve a core human like G2 glycan level over 76% on Host Cell Proteins (HCP).

The G2 glycosylation data was presented by Anne Huuskonen from the VTT Technical Research Centre of Finland Ltd (“VTT”) at the ECFG15 conference in Rome, Italy on Wednesday, February 19. The scientific results are outlined in the presentation entitled, ''Development of filamentous fungus Myceliophthora thermophila C1 into a next-generation therapeutic protein production system.”

A copy of the press release is attached as Exhibit 99.1 and the Presentation is attached as Exhibit 99.2 to this Current Report on Form 8-K herein by reference.

Item 9.01 Financial Statements and Exhibits

(d) Exhibits

The following exhibit is being furnished herein:

Exhibit

Number

Description

99.1

Press Release dated as of February 24, 2020

99.2

VTT presentation entitled "Development of the filamentous fungus Myceliophthora thermophila C1 into a next-generation therapeutic protein production system"

Pursuant to the requirements of the Securities Exchange Act of 1934, as amended, the Registrant has duly caused this report to be signed on its behalf by the undersigned hereunto duly authorized.

Date: February 24, 2020


Dyadic International, Inc.

By:			/s/ Mark A. Emalfarb
Name:			Mark A. Emalfarb
Title:			Chief Executive Officer

DYADIC ANNOUNCES NEW MILESTONE

AS G2 HUMAN LIKE GLYCOSYLATION REACHED

–Further Extends Company’s Biologic Market Opportunities –

JUPITER, FL / ACCESSWIRE / February 21, 2020 Dyadic International, Inc. (“Dyadic”) (NASDAQ: DYAI), a global biotechnology company focused on further improving and applying its proprietary C1 gene expression platform to accelerate development, lower production costs and improve the performance of biologic vaccines, drugs, and other biologic products at flexible commercial scales, announced that data presented at the 15^th European Conference on Fungal Genetics (“ECFG15”) demonstrated that its C1 strain has been glyco-engineered to achieve a core human like G2 glycan level over 76% on Host Cell Proteins (HCP).

https://www.dyadic.com/wp-content/uploads/2020/02/VTT-AHuuskonen-Rome-2-19-2020.pdf

“Our collaboration with Dyadic continues to achieve new scientific milestones regarding glycoengineering Dyadic’s C1 industrially proven cell line, building upon the 95% core human G0 glycosylation milestone that was reported by Markku Saloheimo, Ph.D., Senior Principal Scientist at VTT, during the PEGS Europe (Protein & Antibody Engineering Summit) in November 2019,” said Anne Huuskonen, VTT Senior Scientist.

“Our C1 glycoengineering efforts continue to target our goals in developing C1 cell lines that produce high proportions of human-like glycoforms such as G0, G2, G0F and G2F on heterologous proteins,” said Ronen Tchelet, Ph.D., Dyadic’s Chief Scientific Officer. “In addition to the glycosylation results, VTT also presented data showing that we continue to make excellent progress in reducing the extracellular protease background by fifty (50) times in C1. The elimination of protease activity makes the C1 cell line more efficient, leading to even higher expression levels and lower cost than before.”

“We are very pleased to announce this additional milestone,” Matthew Jones, Dyadic’s Chief Commercial Officer stated, “These important G0 and G2 glycosylation and other scientific advances further demonstrate the power of our C1 gene expression platform coupled with the dedication and world-class expertise of the VTT scientists. We expect that these scientific achievements will open new doors to apply C1 to a broader array of glycosylated biopharmaceuticals, further extend the Company’s market opportunities for biologic vaccines and drugs and continue to generate interest from biotech and pharmaceutical companies, academic and other institutes as well as governmental agencies in animal and human health industries.”

About VTT Technical Research Centre of Finland Ltd

VTT Technical Research Centre of Finland Ltd is one of the leading research and technology organizations in Europe. VTT has a national mandate in Finland. We use our research and knowledge to provide expert services for our domestic and international customers and partners. We serve both private and public sectors. We have 75 years' experience supporting our clients growth with top-level research and science-based results. For more information, please visit www.vttresearch.com.

About Dyadic International

Dyadic International, Inc. is a global biotechnology company which is developing what it believes will be a potentially significant biopharmaceutical gene expression platform based on the fungus Myceliophthora thermophila, named C1. The C1 microorganism, which enables the development and large scale manufacture of low-cost proteins, has the potential to be further developed into a safe and efficient expression system that may help speed up the development, lower production costs and improve the performance of biologic vaccines and drugs at flexible commercial scales. Dyadic is using the C1 technology and other technologies to conduct research, development and commercial activities for the development and manufacturing of human and animal vaccines and drugs (such as virus like particles (VLPs) and antigens), monoclonal antibodies, Fab antibody fragments, Fc-Fusion proteins, biosimilars and/or biobetters, and other therapeutic proteins. Recently, Dyadic has also begun exploring the use of its C1 technology and other technologies to conduct research, development and commercial activities for the development and manufacturing of Adeno-associated viral vectors (AAV), certain metabolites and other biologic products. Dyadic pursues research and development collaborations, licensing arrangements and other commercial opportunities with its partners and collaborators to create synergy by leveraging Dyadic’s technologies in development and manufacture of biopharmaceuticals. In particular, as the aging population grows in developed and undeveloped countries, Dyadic believes the C1 technology may help bring biologic vaccines, drugs and other biologic products to market faster, in greater volumes, at lower cost, and with new properties to drug developers and manufacturers, and as a result improve access and cost to patients and the healthcare system, and most importantly, save lives.

Please visit Dyadic’s website at www.dyadic.com for additional information, including details regarding Dyadic’s plans for its biopharmaceutical business.

Safe Harbor Regarding Forward-Looking Statements

This press release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934, including those regarding Dyadic’s expectations, intentions, strategies and beliefs pertaining to future events or future financial performance. Actual events or results may differ materially from those in the forward-looking statements as a result of various important factors, including those described in Dyadic’s most recent filings with the SEC. Undue reliance should not be placed on the forward-looking statements in this press release, which are based on information available to us on the date hereof. Dyadic assumes no obligation to update publicly any such forward-looking statements, whether as a result of new information, future events or otherwise. For a more complete description of the risks that could cause our actual results to differ from our current expectations, please see the section entitled “Risk Factors” in Dyadic’s annual reports on Form 10-K and quarterly reports on Form 10-Q filed with the SEC, as such factors may be updated from time to time in Dyadic’s periodic filings with the SEC, which are accessible on the SEC’s website at www.dyadic.com.

Dyadic International, Inc.

Chief Executive Officer

Phone: (561) 743-8333

Email: memalfarb@dyadic.com

Development of the filamentous fungus Myceliophthora thermophila C1 into a next-generation therapeutic protein production system Anne Huuskonen VTT Technical Research Centre of Finland Ltd

Myceliophthora thermophila (Thermothelomyces heterothallica) � Thermophilic fungus originally isolated from alkaline soil in Russia � Designated earlier as Chrysosporium lucknowense and currently as Myceliophthora thermophila � Isolated for its ability to produce neutral/alkaline cellulases for use in textile applications � Dyadic Announced Successful Completion of Sequencing of C1 Fungal Genome (May/2005) � Genome sequenced 2011 by JGI (Nat. Biotechnol. 2011 Oct 2;29(10):922-7) � Dyadic International Inc. has developed an industrially proven expression system based on the fungus Myceliophthora thermophila, C1 Expression System � Improved production strains with unique morphology and low viscosity � C1 received a Generally Recognized As Safe (GRAS) designation from the FDA, with no viruses detected, no mycoplasmas, and no detectable mycotoxins (FDA, 2009) � Highest production level of enzymes >120 g/L � Highest production level of an individual recombinant enzyme 80 g/L

Our goal To further develop C1 into efficient gene expression system of biologic vaccines and drugs, to help speed up the development, lower production costs and improve the performance at flexible commercial scales. Efficient Expression Reducing Proteolytic Glycoengineering Activity • Library of promoters, carrier • Generation of humanized proteins, signal sequences and terminators • Identification of key proteases protein-glycan structures • Synthetic Expression System • Deletion of protease genes • Engineering a G0-glycan (SES) • Characterization and utilization producing C1 • Dual vectors of protease deletion strains • Split-marker technology • Marker recycling • Site-specific or random integration

C1 lineage of protease deficient strains DNL104 4xΔ Systematic deletion of protease genes based on: � Isolation and identification of extracellular proteases � C1 protease library in Pichia pastoris DNL110 5xΔ DNL150 13xΔ � More than 50 proteases � Effect of different protease inhibitors on protease activity DNL115 6xΔ DNL145 12xΔ � mRNA sequencing data � Protease gene annotation DNL120 7xΔ DNL140 11xΔ DNL121 7xΔ DNL135 10xΔ DNL125 8xΔ DNL131 9xΔ

Total extracellular protease activity is greatly reduced in C1 protease deficient strains � Direct fluoresence-based assay with casein substrate � The activity of the 12xΔ strain is reduced over 50-fold compared to the 1xΔ strain (fermentation cultures) � The activity of the 13xΔ strain is reduced over 15-fold compared to the 4xΔ strain (MTP cultures) Fermentation culture MTP culture

Protease deletions are improving stability of target proteins Target proteins were spiked into the culture supernatants of the different protease deletion strains ΔprotH-2 ΔprotI-9 ΔprotI-10 ΔprotI-11 ΔprotJ-23 ΔprotJ-16 std Mw 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/2n 0h 3h o/n o/2n 200ng ΔprotH has positive effect on stability Fc-fusionprotein Vaccine antigen 5xΔ 6xΔ 10xΔ 11xΔ 9xΔ 10xΔ 0h 4h o/n 0h 4h o/n 0h 4h o/n o/2n 0h 3h o/n o/2n 0h 3.5h o/n o/2n 0h 3.5h o/n o/2n std Increased stability with more advanced strains Fc-fusion protein: Yellow full-length Red degradation product

Production of Biologics in C1 - Success in expressing high level of ZAPI antigen � Schmallenberg virus antigen coupled with Spytag - difficult-to-express protein in animal cells and microbial systems � First strain had a native C1 promoter in 6xΔ protease strain � Using synthetic promoter (SES) for expression, higher copy number and 8x protease deletion strain increased production six-fold � Development of the protease deficient strain and process conditions increased the amount even further � The C1 produced protein formed efficiently nanoparticles with Spycatcher and showed good immunogenicity in cattle 2000 1830 1800 � The new strain using SES 1600 promoter system in improved 1400 protease deletion background 1200 significantly increased the 1000 production and stability of the 720 800 target antigen 600 Purified antigen (mg/L) antigen Purified 400 120 200 � Up to 1.8 g/L was purified by 0 affinity chromatography 6xΔ 8xΔ 9xΔ

Advantage of C1 over Yeast and CHO � C1 glycan structure is more mammalian like than typically in yeasts � The native C1 glycans are mostly high mannose type (Man3-Man9) including some hybrid glycans � Less engineering steps needed for C1 � Stable genome - defined glycan structure is stable from culture to culture and batch to batch � We aim at defined mammalian glycan forms G0, G0F, G2 and G2F in our glycoengineering efforts Typical Yeast Glycan Structure Dyadic C1 Glycan Structure Targeted Mammalian GlycoformStructures Man30-50 Man3-9 G0 G0F G2 G2F

Two main approaches to gain high Man3 � Man3 is the important precursor of G0 glycans � Two approaches for high Man3: � Deletion of alg3 and over-expression of Mannosidase I � Deletion of alg3 and alg11 Asn Asn Man3 G0 Adapted from Stanley et al., 2008, N- glycans. In: Essentials in Glycobiology. Varki et al, eds.) 9

Two main approaches to gain high Man3 � Man3 is the important precursor of G0 glycans � Two approaches for high Man3: � Deletion of alg3 and over-expression of Mannosidase I ALG3 Deletion � Deletion of alg3 and alg11 Asn Asn Man3 G0 Adapted from Stanley et al., 2008, N- glycans. In: Essentials in Glycobiology. Varki et al, eds.) 10

The effect of alg3 deletion in C1 Wild type strain alg3 deletion strain alg3 deletion strain - native proteins – native proteins – Nivolumab � The alg3 gene was deleted by replacing it with a marker gene � Glycans on native proteins and on purified Nivolumab from shake flask cultures were analyzed � Deletion of alg3 gene significantly simplified the glycan pattern both on native proteins and on Nivolumab � All higher Mw glycans and hybrid glycans were omitted � Substantial amounts of Hex6, Man5 and Man4 glycans remained � Further engineering along this strategy is in progress

Applying alg3-alg11 deletion strategy to produce G0 glycans alg3 deletion strain alg3-alg11 deletion with GNT1+2 alg3 deletion strain alg3-alg11 deletion with GNT1+2 – native proteins - native proteins – Nivolumab - Nivolumab � The alg11 gene was deleted from an alg3 deletion strain. Simultaneously heterologous Glycan Relative GNT1 and GNT2 were expressed from the alg11 locus. amount � Glycan levels with 94% of G0 have been reached. In addition to G0, only Man3 and M3 3.64% GlcNAcMan3 remain in the glycan pattern. M3-GlcNAc 0.45% � G0 glycan levels of over 95% have been reached with this strategy on native proteins. G0 94.11%

The effect of flippase expression No flippase Expression of Expression of overexpression flippase 1 flippase 2 Glycan Area Relative Glycan Area Relative Glycan Area Relative (Abundance) amount (Abundance) amount (Abundance) amount M3 69.34 7.05% M3 116.77 2.84% M3 259.86 3.00% G0 914.85 92.95% M3-GlcNAc 95.17 2.32% M3-GlcNAc 248.09 2.86% Sum of peaks 984.19 100% G0 3894.9 94.84% G0 8162.84 94.14% Sum of peaks 4106.84 100% Sum of peaks 8670.79 100% � Strains were made where two different flippase variants were expressed in addition to GNT1 and GNT2 in alg3-alg11 deletion background � The total glycosylation level (sum of peaks) in shake flask cultures increased ~4x (flippase 1) or ~9x (flippase 2) � The glycan pattern remained good with 93-95% G0 glycans on native proteins

Towards G2 glycans on target proteins alg3-alg11 deletion with GNT1+2 G0 strain with GalT - native proteins - native proteins Glycan Relative amount M3 3.16% M3-GlcNAc 0.42% G0 5.81% G1 6.97% G1 5.53% G2 76.38% � The human GalT1 gene was expressed in a strain with G0 glycan machinery � Glycans on native proteins from shake flask culture are shown � G2 glycan levels over 76% have been reached with this strategy. In addition to G2, over 10% of G1 glycan forms and some residual G0 and Man3 were detected.

Summary � Myceliophthora thermophila C1 is an industrialized protein Product Production level production host that is now developed for therapeutic g/L protein manufacture – with several large biopharma companies entering into collaborations. Mabs 24.5 � We have identified critical proteases to deal with for Fabs 14.5 therapeutic protein production enhancement and enabled a very significant reduction of the protease load in the Fc-fusion proteins 13.2 production strains. Difficult-to-express � Our glycoengineering program aims at high proportions of human glycoforms G0, G2, FG0 and FG2. Excellent G0 Bispecific antibodies 1.0 levels have been reached through alg3-alg11 deletion Trispecific nanobody 6.6 strategy. � Monoclonal antibodies have been produced in C1 with Viral antigens 1.8 levels reaching 24.5 g/l and rates up to 3.5 g/l/day. Fab VLPs 2.2 fragments have been expressed at levels up to 14.5 g/l and Fc-fusion proteins up to 13.2 g/l in a 7-day process. (extracellular prod.) � Difficult-to-express proteins have been produced in C1 at Bacterial vaccine 6.0 superior levels as compared with other production systems. protein

Acknowledgements VTT Protein Production Dyadic International Inc. Anne Huuskonen Ronen Tchelet Marika Vitikainen Gabor Keresztes Georg Schmidt Mark A. Emalfarb Marilyn Wiebe Veera Korja Anssi Rantasalo VTT Analytics VTT Biosensors Andriy Kovalchuk Heli Nygren Kristina Iljin Kari Koivuranta Natalia Maiorova Outi Koivistoinen Tino Koponen Hanna Kuusinen Kaisa Roine LC ESI MS/MS Karita Viita-aho Turku Proteomics Facility, Merja Aarnio University of Turku and Åbo Akademi University Sirpa Holm Taru Westerholm Christopher Landowski Contact Dyadic – rtchelet@dyadic.com

THANK YOU! rtchelet@dyadic.com anne.huuskonen@vtt.fi