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

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.) ALG11 Deletion 11

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