The use of in silico analysis to engineer the best immunogenic epitope and produce the corresponding prophylactic antigen-based vaccines with C1 production platform in order to rapidly respond to viral pandemics

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The C1 gene expression system, disrupting the way biologic vaccines, and drugs are developed & manufactured

Dyadic International, Inc. is a global biotechnology company which is developing what it believes will be a potentially significant biopharmaceutical protein production system based on the fungus Myceliophthora thermophila, nicknamed 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, production and 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, monoclonal antibodies, biosimilars and/or biobetters, and other therapeutic proteins. Dyadic pursues research & development collaborations, licensing arrangements and other commercial opportunities with its partners and collaborators to leverage the value and benefits of these technologies in developing and manufacturing biopharmaceuticals which these technologies help produce. In particular, as the aging population grows in developed and undeveloped countries, Dyadic believes the C1 technology may help bring biologic drugs to market faster, in greater volumes, at lower cost, and with new properties to drug developers and manufacturers and, hopefully, improve access and cost to patients and the healthcare system, but most importantly save lives

The use of in silico analysis to engineer the best immunogenic epitope and produce the corresponding prophylactic antigen-based vaccines with C1 production platform in order to rapidly respond to viral pandemics

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C1: How the C1 platform will change the production approach for therapeutic proteins

For over 30 years, Dyadic has proven itself, both commercially and scientifically, to be a high quality and highly productive producer of enzymes and proteins for specialty chemical applications using a proprietary and patented expression system based on the Myceliopthora thermophila fungus, nicknamed C1. The C1 platform technology, a hyper-productive fungal expression system, was used to develop & manufacture large quantities of desired proteins at industrial scale at significantly lower capital and operating expenditures. In this presentation we shall demonstrate how the benefits of C1 as a successful production host is now being harnessed by Dyadic to produce biological medicines and vaccines. Using new and improved C1 base strains along with better molecular genetics tools that have been developed over the past several years, we demonstrate the ability of C1 to express mAbs that are secreted, folded correctly and reach high yields. MAbs produced in C1 have almost identical binding kinetics to mAbs produced using CHO cells. In addition, our research program includes comprehensive approach to identify and knock-out proteases for further enhancing protein stability and improving yields. We have also achieved encouraging results, knowledge and experience in the rVaccine space from our prior research collaboration with Sanofi Pasteur to express rVaccines against Influenza virus. Results of a mice test that was conducted by Sanofi Pasteur, clearly demonstrated that HA produced by C1, generated high immunogenicity response against the virus without any adverse affects. Like other filamentous fungal strains, C1 has high mannose glycoform structures. However, unlike most fungi and yeasts, C1 does not have ‘high’ mannose (branched 30-50 mannose species), but rather has ‘oligo’ mannose structure (branched 5-9 mannose species). In addition, no O-glycosylation has been observed on C1 secreted proteins, in contrast to Pichia that O-glycosylates all secreted antibodies, necessitating deletion of the O-glycosylation machinery. Using the benefits of those advantages, we have started Glycoengineering program aiming to develop C1 strain that produces proteins with defined human-like glycan patterns. The progress we have already made in C1 glycoengineering will also be presented. Thus, Dyadic firmly believe that the C1 strains that we are developing with offer certain competitive advantages over other leading pharmaceutical expression systems, such as CHO cells, has the potential to become the production system of choice for therapeutic protein and vaccines manufacturing

Affinity purification of SARS-COV-2 spike protein receptor binding domain produced in a C1 fungal expression system

The Receptor Binding Domain (RBD)of the spike protein of SARS-CoV-2 has shown promise for diagnosis, treatment, and development of vaccines for COVID-19. However, two problems persist with large scale production of RBD: 1) lack of high productivity upstream cell culture, 2) absence of a commercial, highly selective affinity resin. In an effort to overcome these limitations, we evaluated two novel technologies for the production and purification of RBD. Briefly, RBD was expressed using C1, an engineered fungal strain of Thermothelomyces heterothallica (DyadicInternational1). The C1 platform expresses glycosylated antigens with high productivity, stability, and purity. RBD was purified using a novel affinity resin2 known to produce yields of 90% to 95% purity in one chromatography step. Affinity purification did not affect protein quality, as demonstrated by ACE-2 binding of RBD. The novel affinity resin showed excellent base stability, consistent product quality, and similar ACE-2 binding activity over 40 cycles. Please click Download on the upper right corner to see the full abstract

Development of the filamentous fungus Thermothelomyces heterothallica C1 into a next-generation production platform for human and animal vaccines

engineering. The thermophilic fungus Thermothelomyces heterothallica is a robust and versatile fungal expression system for the rapid production of proteins at very high levels. In the last 6 years, the C1 protein production platform has been further improved to become a safe and efficient expression system with the prime objective of speeding up the development and production of commercial scale human and animal vaccines, monoclonal antibodies, biosimilars, as well as other therapeutic proteins at larger quantities and lower cost. C1 is a very efficient platform to produce antigens, even to generate multicomponent vaccines. The production levels of engineered C1 strains are similar in terms of yield and purity, reaching in some cases more than 2.5 g/L (in 4-5 days). In contrast to other vaccine platforms, C1 has a higher safety profile, and production can be scaled up in a more cost-effective manner using standard microbial E. coli fermenters. Stable cell lines have been developed to produce different antigens as influenza, neuraminidase, west Nile, rabies, rift valley fever..etc. Please click Download on the upper right corner to see the full abstract

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1

INTRODUCTION: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. METHODS: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. RESULTS: One dose of 10-μg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. DISCUSSION: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system

Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine

Toxicity and Local Tolerance of a Novel Spike Protein RBD Vaccine Against SARS-CoV-2, Produced Using the C1 Thermothelomyces Heterothallica Protein Expression Platform

Coronavirus disease 2019 (COVID-19) has caused the ongoing COVID-19 pandemic and there is a growing demand for safe and effective vaccines. The thermophilic Thermothelomyces heterothallica filamentous fungal host, C1-cell, can be utilized as an expression platform for the rapid production of large quantities of antigens for developing vaccines. The aim of this study was to evaluate the local tolerance and the systemic toxicity of a C1-cell expressed receptor-binding domain (C1-RBD) vaccine, following repeated weekly intramuscular injections (total of 4 administrations), in New Zealand White rabbits. The animals were sacrificed either 3 days or 3 weeks following the last dose. No signs of toxicity were observed, including no injection site reactions. ELISA studies revealed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G antibodies in the sera of C1-RBD-treated animals starting from day 13 post injection, that were further elevated. Histopathology evaluation and immunohistochemical staining revealed follicular hyperplasia, consisting of B-cell type, in the spleen and inguinal lymph nodes of the treated animals that were sustained throughout the recovery phase. No local or systemic toxicity was observed. In conclusion, the SARS-CoV-2 C1-RBD vaccine candidate demonstrated an excellent safety profile and a lasting immunogenic response against receptor-binding domain, thus supporting its further development for use in humans