DYAI: Glycosylation Milestone Opens Doors For mAb Production

By John Vandermosten, CFA

NASDAQ::DYAI

Dyadic DYAI achieved an impressive milestone in its glycoengineering efforts which was announced via VTT's¹ presentation at the Protein and Antibody Engineering Summit (PEGS) Europe in Lisbon a few weeks ago. The event was memorialized in a press release on November 25th. It announced that Dyadic's filamentous fungi expression system Myceliophthora thermophila, or more simply C1, has been able to produce the core human-like G0 glycan structure. G0 provides the basic building block for pursuing more complex forms of carbohydrate chains such as G0F, G2 and G2F. These forms are critical for therapeutic efficacy, immunogenicity, protein stability, moderation of half-life of proteins and optimization of biologics, such as monoclonal antibodies like nivolumab which Dyadic is currently seeking to produce.

Exhibit I – Targeted Mammalian Glycoform Structures²

Glycosylation is an important post-translational modification that attaches a carbohydrate (sugar molecule) to a protein. The structure provides an array of benefits that affect protein structure, function and stability and protein folding. Glycosylation determines the activity of amino acid chains when bound to receptors and alters how they recruit, interact and activate signaling proteins. Glycans that appear on monoclonal antibody or Fc-fusion proteins have a material impact on the pharmacokinetics and pharmacodynamics of these biologics. We previously discussed glycosylation in a brief supplement available here.

The cells of non-human species do not glycosylate proteins the same way human cells do and using these cells to produce biologics for human use requires the application of synthetic biology to achieve the correct form. Improperly glycosylated therapeutic proteins can be immunogenic and cause adverse reactions in patients or may fail to have the desired effect. This is because the glycans which appear on the membrane of a cell label them as either self or foreign. If the proteins are seen as self, then therapeutic biologics that present the proper glycoforms can interact with the immune system in the intended way.

Many therapeutic proteins require mammalian glycosylation; a post translational modification that is generally not possible with bacterial, yeast, insect or viral expression systems. Chinese hamster ovary (CHO), mouse myeloma and human embryonic kidney (HEK) cells are frequently used as host of choice for recombinant protein therapeutics that require human glycoforms. However, these systems have several shortcomings that limit their efficient use such as variable consistency, low productivity, costly media and the requirement for suspension cultures.

Many of the top selling biologics require human glycosylation and are reliant on the CHO system to provide it. Some examples of these leading products includes Rituxan (G0, G1 & G2), Remicade (G0, G1 & G2), Avastin (G0 & G1) and Herceptin (G0 & G1) to name a few. C1's ability to express these biologics with the proper glycoforms in an efficient and cost effective manner should leave the leading biotech companies that are reliant on CHO deeply concerned. We see the achievement of G0 as yet another incentive for large biologics developers to partner with Dyadic.

In the recent presentation by VTT referenced above, yields of 92% to 95% for G0 were achieved using a variety of approaches. The most successful of these efforts selected the expression of flippase 1 and the deletion of the alg3 and alg11 gene to produce a G0 yield of 95%. This high yield achievement increases the diversity, breadth and scope of products Dyadic can make. G0 is the base on which C1 Dyadic can pursue G0F, G2, G2F and others that confer specific features to proteins that are coated with these complex sugars.

Exhibit II – Concentration of G0 Under Various Approaches³

Dyadic employed two approaches in the lab to increase the output of Man3⁴, which is an important precursor to G0. The first approach was the deletion of alg3⁵ and over-expression of mannosidase I and the second was deletion of alg3 and alg11⁶. See Exhibit III. In the first approach the alg3 gene was replaced with a marker gene and the resulting glycan pattern was simplified. Higher molecular weight glycans and hybrid glycans were omitted and material levels of Hex6, Man5 and Man4 glycans remained. The second approach deleted the alg11 gene from the alg3 deletion strain. Heterologous GNT1⁷ and GNT2⁸ were simultaneously expressed from the alg11 locus. This was the most successful approach, producing G0 glycan levels up to 95%. The only other constituents in the purity count were GlcNAcMan3 and Man3 at 2% and 3% respectively.

There is also a third and more complicated approach to achieve G0 which requires expression of mannosidase I and GNT2. Dyadic is not presently pursuing this route but it remains an option. Below we show the pathway for proteins as they transit the endoplasmic reticulum (ER). The proteins begin their journey on the cytosolic surface of the ER and pass through multiple enzymatic steps. After several steps, the oligosaccharide is translocated from the cytosolic surface across the membrane to the ER lumen assisted by flippase making it available to enzymes in the lumen.

Exhibit III – Enzymatic Pathway for Glycosylated Proteins Transiting the ER⁹

Now that Dyadic has successfully produced the G0 glycoform on expressed proteins, the focus is on achieving a higher output volume. The company will also pursue higher order glycostructures such as G0F, G2 and G2F which add galactose and fucose to the chain. C1 also has the flexibility to produce structures that are attractive to animal health, such as Man9. With the high 95% yield of G0, Dyadic can provide a broader product offering that may be superior to existing biologics due to lower immunogenicity from a purer glycoform profile. We are very encouraged by Dyadic's achievement of this important milestone along with parallel advancements in protease deletion and high output that make C1 a radically disruptive technology.

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1. VTT is the Technical Research Center of Finland, a state owned and non-profit company.

2. Source: Novel Highly Productive Production System for Biotherapeutics: Filamentous Fungus Myceliophthora thermophila C1. VTT Technical Research Centre of Finland Ltd. November 21, 2019. Note: Colored shapes are monosaccharides. Blue square is N-Acetylglucosamine, green circle mannose, red triangle fucose and yellow circle galactose.

3. Source: Novel Highly Productive Production System for Biotherapeutics: Filamentous Fungus Myceliophthora thermophila C1. VTT Technical Research Centre of Finland Ltd. November 21, 2019.

4. Man3 is the addition of three mannose sugar chains to the N-Acetylglucosamine base.

5. ALG3 is a gene that encodes mannose in the glycosylation process.

6. ALG11 is a gene that encodes mannose in the glycosylation process.

7. Glucose N-acetyltransferase 1 protein

8. Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase protein

9. Source: Novel Highly Productive Production System for Biotherapeutics: Filamentous Fungus Myceliophthora thermophila C1. VTT Technical Research Centre of Finland Ltd. November 21, 2019. Adapted from Stanley et al., 2008, N-glycans. Essentials in Glycobiology. Varki et al.