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PROGRAM CHAIRS:
Manuel J.T. Carrondo, PhD - Instituto de Biologia Experimental e Tecnológica (iBET)
Clifton E. McPherson, PhD - Protein Sciences Corporation, A Sanofi Company


Dominic Esposito, PhD
Director, Protein Expression Laboratory
Frederick National Laboratory for Cancer Research
and Yu Fu
PhD Student
University of Massachusetts Medical School
The Complete Genome Sequences of Two Trichoplusia ni Cell Lines

Abstract

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest, Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. This genome assembly contains 2.92 Mb of highly repetitive W chromosome, providing insights into W chromosomes in Lepidoptera. We found that Hi5 cells produce abundant siRNAs mapping to a positive-sense, bipartite alphanodavirus (TNCL virus), indicating Hi5 cells mount an RNAi defense to the TNCL virus. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. The T. ni genome provides insights into pest control and allows Hi5 cells to be genetically manipulated to facilitate recombinant protein production.

Biographies

Dr. Esposito is currently the Director of the Protein Expression Laboratory (PEL) at the Frederick National Laboratory for Cancer Research (FNL) in Maryland. The PEL primarily generates DNA and protein reagents for the NCI national mission to target KRAS-driven cancers, as well as supporting NCI and NIH intramural investigators in production of difficult proteins. Prior to his role as director, Dr. Esposito led the Clone Optimization Group in the PEL for nine years and was responsible for the generation of over 15,000 expression clones, 400 new expression vectors, and several technological innovations in protein expression. Dr. Esposito received his BA in Chemistry at La Salle University in Philadelphia, and his PhD in Biochemistry at the Johns Hopkins University Bloomberg School. Dr. Esposito previously worked for Life Technologies, where he helped to develop the Gateway recombinational cloning system.

Yu Fu is a PhD student in the Bioinformatics Program at Boston University. He did his research at University of Massachusetts Medical School with Prof. Zhiping Weng and Prof. Phillip Zamore. His research interests include computational analysis of small silencing RNAs, and developing bioinformatics pipelines for rapid data processing.

António M. Roldao, PhD
Senior Scientist, Instituto de Biologia Experimental e Tecnológica (iBET)
Bioprocess Engineering of Insect Cells for Pseudotyped VLP Expression and Optimization

Abstract

Conformational-complex membrane proteins (MPs) are vaccine/drug targets in many diseases, but drug and vaccine development has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of ordered nanoscale architectures such as virus-like particles (VLPs), preserving their native lipidic environment.

In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect cell platforms for fast production of enveloped VLPs pseudo-typed with target MPs. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept (PoC) demonstrated using two membrane proteins, the influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes were designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing improvement of HIV Gag-VLP production in the developed stable insect cells. Under hypothermic culture conditions, adapted cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the latter alone leads to lower production yields. A more modest increase in productivity (up to seven-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. PoC was successfully demonstrated in 0.5 L stirred-tank bioreactors.

Profiting from the platforms developed above, a modular system comprising stable and baculovirus-mediated expression in insect cells was established for the production of a multi-HA influenza VLP as vaccine candidate that otherwise could not be obtained due to baculovirus vector instability. By combining stable with transient expression systems, we could rationally distribute the number of genes to be expressed per platform and thus generate the target VLP for subsequent animal studies. In addition, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth resulting in a four-fold increase in HA titers per mL. PoC was successfully demonstrated in 2 L stirred-tank bioreactors. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate drug and vaccine development.

Biography

António Roldao is a Chemical Engineer with a PhD in Engineering and Technology Sciences - Systems Biology (2010) from ITQB-UNL (Portugal). From 2010 to 2014 he was a senior researcher and director of fermentation technology at SysBio group headed by Dr. Jens Nielsen at Chalmers University of Technology (Sweden). In 2014 he became a senior researcher at the Animal Cell Technology Unit headed by Prof. Paula Alves and Prof. Manuel Carrondo at iBET / ITQB-NOVA (Portugal), and since 2015 he has been Investigador FCT. António has been awarded with several research grants, is an author of 18 scientific manuscripts in peer-reviewed journals, author or co-author of seven book chapters/conference proceedings, and has given over 25 oral and poster communications. António has been involved in many Portuguese and EU funded research projects. Current research focuses on the development of novel complex biologics with an impact in Human Health, e.g. VLP-based vaccines against infectious diseases such as influenza and dengue. To accomplish such objectives, bioprocess engineering and bottom-up systems biology approaches are combined with process monitoring and product characterization, thus undoubtedly fastening the generation of such products.

Christoph Geisler, PhD
Chief Research Scientist, GlycoBac LLC
Adventitious Viruses Contaminating Insect Cell Lines

Abstract

Starting in the late 1970s, a wide variety of viruses had been found to contaminate insect cell lines. Surprisingly, adventitious viruses contaminating insect cell lines routinely used for recombinant protein and AAV production were not discovered until 2007 (High Five™) and 2014 (Sf9). These discoveries raised important questions regarding the biosafety of therapeutics produced in this platform. In this talk, I will discuss viruses that can contaminate insect cell lines, including several relatively little-known ones. As most persistent viral infections of insect cell lines were discovered serendipitously, I will also discuss how these viruses were discovered as well as detection methods that can be used to probe for viral contamination. Finally, I will present new virus-free insect cell lines that have been developed as alternatives to contaminated cell lines, and to what extent these can serve as viable alternatives to their well-established, contaminated counterparts.

Biography

Christoph Geisler has worked in the baculovirus insect cell system since 2005. He has experience in both cell line development and baculoviral vector engineering, and specializes in the glycobiology of the system. Recently, he has developed new bioinformatics approaches to screen for adventitious viral contaminants in cell lines used to produce biopharmaceuticals.

Kendra Steele, PhD
Senior Research Scientist, ParaTechs Corporation
Simplifying Membrane Protein Purification: Introducing a Fluorescent BEVS Protein System that Enhances Protein Production and Greatly Simplifies Detergent Screening

Abstract

Membrane proteins are central and essential to cellular structure, metabolism, and function. Consequently, membrane protein mutations frequently cause disease and are often therapeutic targets. Over 60% of today’s drug targets are membrane proteins with 30% of those treatments targeting G-protein coupled receptors (GPCRs), an important family of cell surface proteins. Despite enormous growth potential and advances in crystallography, mass spectrometry, and protein-ligand interactions, GPCR therapeutics remain difficult to develop, largely because GPCRs are difficult to isolate in intact and functional forms. Purification methods are often cumbersome, time-consuming, and ineffectual, hampering GPCR purification and downstream experimentation. Thus, rapid and simplified methods that can be tailored for purification of specific membrane proteins would greatly enhance opportunities in this important therapeutic sector.

The challenge to purifying membrane proteins is two-fold: to produce large quantities of pure, properly processed protein, and to solubilize active protein from membranes. Mammalian membrane proteins, and notably GPCRs, are often successfully produced with the baculovirus expression vector system (BEVS) in insect cells. ParaTechs has improved the BEVS by introducing viral ankyrin proteins (vankyrins) that delay apoptosis of baculovirus-infected insect cells, thereby enabling prolonged synthesis, accumulation, and processing of complex recombinant proteins. But the ability to solubilize and isolate the membrane protein in a native, functional form is perhaps even more important than achieving a high level of protein production. A major impediment to membrane protein research is simply finding the right detergent and conditions to purify your protein. Identifying the right detergent typically begins with an uninformed screen of detergents selected with inadequate knowledge of the proteins physio-chemical properties. Commercially available detergent panels do not systematically manage membrane protein purification, isolation, and analyses. Kit users are required to develop, process, and analyze each sample by methods unspecified by the kit. For example, if a user purchases a kit with 96 detergents, they must perform 11 Western blots to screen each sample (if using a 10-lane polyacrylamide gel with molecular weight marker). Here, we introduce VELucity, a fluorescent protein system that enhances production, improves stability, and enables screening for solubilization of proteins in a panel of commercial detergents before protein purification. This system will allow researchers to rapidly determine the right detergents for their protein by visual fluorescence, thereby reducing screening time from days to minutes.

Biography

Dr. Steele is the lead scientist on baculovirus protein expression projects at ParaTechs Corporation. She is the creator of VELucity, a fluorescent protein system that enhances membrane protein production, improves stability, and enables screening for solubilization of proteins in a panel of commercial detergents before protein purification. She also runs the contract program that expresses a user’s protein using the vankyrin enhanced-baculovirus expression system.

Eric Horowitz, PhD
Associate Director, Analytical Development and QC, Voyager Therapeutics
Biochemical and Biophysical Characterization of Adeno-Associated Viruses Produced by Triple Transfection in HEK293 Cells and with Insect Cells/Baculovirus Expression System

Abstract

In this presentation, there will be shown biochemical, biophysical, and bioassay data for the same AAV vectors produced with the two different expression systems. Implications for AAV development and manufacturing for gene therapy applications will be discussed as well.

Biography

Eric Horowitz is the head of analytical development and quality control at Voyager Therapeutics. Eric has a PhD in analytical chemistry from Georgia Tech and did his postdoctoral work in AAV biology and biophysics at University of North Carolina at Chapel Hill with Prof. Aravind Asokan.

Gorben P. Pijlman, PhD
Associate Professor Arbovirology, Laboratory of Virology, Wageningen University
Tweaking Baculovirus Expression Vectors to Stabilize Transgenes and Polish Enveloped VLP Vaccines

Abstract

The baculovirus-insect cell expression system is an ideal platform to express glycoproteins and enveloped virus-like particles (eVLPs) of medically important arboviruses such as chikungunya, West Nile, and Zika. Arboviruses actively replicate in mosquitoes and use these as transmission vectors. Large-scale or continuous protein production by recombinant baculoviruses suffers from yield loss, partly due to transgene instability in bacmid-based expression vectors. By relocating the att-Tn7 transposition site on the bacmid, we have identified a locus that combines high level expression with improved transgene stability. We now use this ‘stabilized’ AcMNPV bacmid as a backbone to express arbovirus glycoproteins and eVLPs for use in diagnostic assays and as vaccines. Recently, we have produced secreted Zika eVLPs and analysed the infection dynamics in a small-scale bioreactor configuration. Electron microscopy analysis demonstrates the production of subviral particles (SVPs, ~30 nm) and eVLPs (~50 nm), which are currently evaluated in a mouse vaccination-challenge experiment. Finally, we have developed a novel method to rapidly clear baculovirus particle contaminants from eVLP preparations, which will aid the further development of baculovirus-expressed eVLPs as effective vaccines for human use.

Biography

Gorben Pijlman obtained his MSc in Biotechnology and Bioprocess Engineering at Wageningen University, the Netherlands in 1999. His PhD research with Professor Just Vlak focused on the molecular mechanisms underlying the ‘baculovirus defective interference’ phenomenon. In 2003, he spent four postdoctoral years at the University of Queensland in Brisbane, Australia, for research on West Nile virus replication, pathogenesis, and non-coding viral RNA. He was a consultant for the Australian biotech start-up RepliKUN to develop flavivirus replicon vectors for vaccines and cancer gene therapy applications. In 2007, he returned to Wageningen to become Associate Professor Arbovirology. The arbovirus research programme is an interesting mix of fundamental virology focused at arbovirus-host interactions using live mosquitoes as a model, and applied studies on next-generation arbovirus vaccines based on baculovirus-expressed VLPs and self-amplifying mRNAs (replicons). His favourite viruses are West Nile, Zika, Usutu, chikungunya, and the enigmatic salmonid alphavirus.

Christopher W. Kemp, PhD
President, Kempbio, Inc.
PEI-Mediated Transient Transfection of Insect Cells for the
Expression of Recombinant Proteins in Stirred-Tank Bioreactors

Abstract

The baculovirus-mediated production of recombinant proteins in insect cells is a mature and robust platform. However, for certain applications, the presence of baculovirus in downstream process fluids presents numerous challenges to the successful purification of the protein of interest. The availability of expression vectors designed to support the transient transfection of insect cells provides a potential alternative to the baculovirus expression vector system (BEVS) in applications where baculovirus removal is an issue. In addition, transient transfection of expression vectors may be a more efficient way of screening multiple constructs in insect cell expression systems than the production of multiple baculovirus constructs. We have developed two transient insect cell expression vectors expressing either SEAP or a particulate antigen and used them to investigate the expression performance of this system in SF9 and High Five cells. Transient transfections were performed using PEI Max in shake-flask and stirred-tank bioreactors under varying conditions of cell density, temperature, and length of incubation. The results suggest that transient transfection of insect cells may be used to produce significant amounts of protein in a rapid and baculovirus-free manner.

Biography

Chris Kemp is President of Kempbio, Inc., a contract protein expression company located in Frederick, Maryland. Dr. Kemp has over 25 years of experience in the scale-up of recombinant protein expression systems including baculovirus, mammalian, and BacMam expression platforms. Chris received his PhD from George Washington University in Washington, DC. He was employed for ten years at the NIH, and for seven years at BioWhittaker in Walkersville, Maryland (currently Lonza). Chris founded the protein expression service company Kemp Biotechnologies, Inc. in 1992 and the molecular biology product company GeneChoice in 2000. The current company, Kempbio was started in 2009 and is focused on rIgG expression, viral glycoprotein production and purification, and the production and purification of virus-like particles (VLPs).

Robin Levis, PhD
Deputy Director, Division of Viral Products, FDA CBER
and
Gabriel I. Parra, PhD
Principal Investigator, Division of Viral Products, FDA CBER
Regulatory Updates and Using Virus-Like Particles to Inform Norovirus Vaccine Design

Abstract

This dual presentation will include: regulatory updates on available licensure pathways for baculovirus-related vaccine products; and a presentation on scientific work related to using baculovirus-expressed virus-like particles (VLPs) for studying the genetic diversity of norovirus strains and vaccine design. With regards to regulatory updates, CBER has been confronted with several disease outbreaks in recent years that have required the consideration of several licensure pathways and expedited review programs to facilitate the development and licensure of vaccines to prevent serious disease. One of the critical lessons learned from these activities has been the importance of collaborative review efforts that include industry, regulators, and other public health agencies. Information on review pathways will be presented and some examples of how these pathways have been utilized will be described. With regards to the use of VLPs to inform norovirus vaccine design, we will discuss how VLPs have played a pivotal role to our understanding of immunity and the antigenic characterization of noroviruses. Some of the most relevant information for norovirus vaccine design that we have learned using VLPs is that: (i) despite the detection of high levels of cross-reactive antibodies, individuals can undergo multiple norovirus infections throughout life, suggesting major antigenic differences among noroviruses; (ii) norovirus capsid protein (VP1) binds to carbohydrates from the human ABH histo-blood group that could act as attachment factors that enhance infection; (iii) antibody-mediated blockade of ABH carbohydrates have been shown to be the best predictor of protection to human norovirus diseases; (iv) the major antigenic sites involved on the ABH carbohydrate blockade map on the most exposed and variable region of VP1; and (v) changes on the major antigenic sites have been linked to emergence of new pandemic strains. Thus, in the absence of a robust cell culture system for human noroviruses, VLPs have served as an important source of antigen to study immune responses and vaccine design against noroviruses.

Biographies

Dr. Levis has worked at the US Food and Drug Administration (FDA) since 1995. She is currently the Deputy Director of the Division of Viral Products in the Office of Vaccines Research and Review at CBER/FDA, a position she has held since 2006. Prior to this position, she served as the Regulatory Coordinator for the Division of Viral Products (2002–2006) and served as a Senior Staff Fellow in the Laboratory of Vector Borne Viral Diseases (1995–2002). Her initial research work at the FDA related to dengue virus replication. She then transitioned to being the lead CMC reviewer for licensed rabies virus vaccine products and rabies vaccine and related products under development. In addition to her work on rabies, Dr. Levis served as the primary CMC reviewer for the Human Papillomavirus (HPV) vaccines to prevent cervical and related cancers. She was the chair of the licensing committee for the review and approval of Cervarix, an HPV vaccine made using the baculovirus expression system to produce viral antigens. Dr. Levis continues to be involved in the review and regulation of all baculovirus-expressed antigens.

Gabriel Parra is currently a Principal Investigator at the Division of Viral Products (DVP), US FDA. His major research interest focuses on the role of viral diversity to overcome immune responses mounted against two gastrointestinal viruses, rotavirus and norovirus. Before joining DVP, he did his postdoctoral studies at the NIAID, NIH working in the development of different tools and reagents, including virus-like particles, to characterize the antigenic properties of human noroviruses.

Arifa S. Khan, PhD
Supervisory Microbiologist, FDA CBER
Progress on the Genome of the Spodoptera frugiperda Sf9 Cell Line

Abstract

We have been evaluating new technologies for the detection of adventitious viruses in vaccine cell substrates. Next-generation sequencing (NGS) and degenerate PCR resulted in our discovery of a novel rhabdovirus in Sf9 cells, which has subsequently been found by others. We found limitations using short-read NGS platforms for investigating endogenous retroviral sequences that may be associated with the reverse transcriptase activity produced from Sf9 cells. Therefore, we have used long-read PacBio sequence technology to obtain the draft whole genome assembly to characterize endogenous retroviral and other endogenous viral sequences in Sf9 cells. This presentation will describe our approach and bioinformatics pipeline for detection of viral sequences and include other ongoing efforts including NGS analysis of a rhabdovirus-free cell clone derived from the Sf9 cell line.

Biography

Dr. Khan is a Senior Investigator in the Office of Vaccines Research and Review in the Center for Biologics Evaluation and Research, US Food and Drug Administration. She joined the FDA in 1991, after working at the National Institute of Allergy and Infectious Diseases (NIAID) in the National Institutes of Health (NIH) since 1979, where she made important contributions in the discovery and characterization of endogenous retroviruses, pathogenic murine leukemia retroviruses, and simian immunodeficiency viruses. Dr. Khan’s current research efforts in CBER are directed toward investigating the use of advanced virus detection technologies for cell substrates and vaccine safety, and evaluating monkey models for preclinical testing of AIDS vaccines. Her regulatory responsibilities include review of Investigational New Drugs and Biological License Applications for a variety of candidate vaccines, including influenza and HIV, and novel cell substrates. Dr. Khan has been involved in the licensure of several viral vaccines and the development of various FDA, International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Public Health Service (PHS), and World Health Organization (WHO) guidance documents related to cell substrates, vaccines, therapeutics, and xenotransplantation. She is a lead in various working groups on advanced virus detection technologies and cell substrate safety. Dr. Khan received her PhD in Microbiology from the George Washington University, Washington, DC.

Scot Shepard
Director, Biologics Process Design, Takeda Pharmaceuticals U.S.A., Inc.
Enabling Access to a Norovirus Virus-Like Particle-Based Vaccine
Through Advanced Manufacturing Process Design

Abstract

With an estimated 699 million cases and 200,000 deaths annually, including 130 million cases of norovirus-related illness in industrialized countries, norovirus is the world’s leading cause of sporadic acute gastroenteritis, with the highest rates of severe disease occurring in young children and older adults. Globally, norovirus infection leads to a total of $4.2 billion in direct health system costs and $60.3 billion in societal costs per year.

Takeda Vaccines is developing a prophylactic vaccine candidate designed to protect against norovirus-related disease. Advanced manufacturing process design concepts have been applied to ensure reliable, affordable global access to the vaccine. The process design combines high titer expression of virus-like particles with orthogonal, high resolution purification methods and advanced analytical characterization. The manufacturing process employs a closed, single-use manufacturing platform that is designed to be portable and compatible with a range of multi-use biomanufacturing facilities. The presentation will provide an overview of the manufacturing process and will summarize a technical transfer project that demonstrates how the process fits into an existing manufacturing facility without the need for process changes or significant facility remodeling.

Biography

Scot Shepard is Director, Biologics Process Design for Takeda Vaccines. Scot has more than 20 years of industry experience including roles in process development, manufacturing, and cross-functional CMC Team management at Takeda Vaccines, Amgen, Bayer, and Diosynth Biotechnologies.

Marc G. Aucoin, PhD
Associate Professor, Center for Bioengineering and Biotechnology
University of Waterloo
Control Using BEVS: From Promoters to CRISPR

Abstract

The insect cell – baculovirus expression vector system (IC-BEVS) has proven to be a robust and efficient platform for the manufacture of recombinant proteins for research and industry. Nevertheless, IC-BEVS suffers from a general lack of developed genetic tools to permit rational genetic engineering to further improve efficiency and yields; the polh and p10 promoters are employed almost exclusively for protein production, and co-infection strategies are still generally employed for the production of multiple heterologous proteins. In these strategies, multiplicity of infection (MOI) and time of infection (TOI) are the parameters used to modulate the relative ratios of recombinant proteins. However, maintaining multiple recombinant BEVs (rBEVs) is tedious and time consuming, and the polh promoter, though capable of driving an immense amount of transcription, is active very late in the infection cycle when host cells may be severely compromised. Moreover, replication of the baculovirus is an inherent process of the infection cycle and may represent a significant diversion of cellular resources away from recombinant protein production. In order to further develop the IC-BEVS as an important commercial-scale production host, these limitations must be addressed.

We have previously demonstrated control over the relative ratios of two recombinant proteins in a co-expression rBEV, in which two fluorescent proteins were encoded and produced by a single rBEV. Modulation of the relative ratios of each fluorescent protein was achieved by employing different promoter combinations to drive expression of each gene. We extended this approach to the production of influenza A virus-like particles and are currently investigating ways to improve yield, and understand the effects on particle morphology.

To address baculovirus replication, we have developed and applied the CRISPR-Cas9 system for transcriptional repression. We have developed an Sf9 cell line constitutively expressing the dcas9 gene and have successfully down-regulated the expression of a fluorescent reporter protein using transfection-based assays in this cell line. We are currently transferring this system to achieve transcriptional repression of heterologous genes delivered by a rBEV as well as endogenous genes encoded by AcMNPV. Our aim is to target AcMNPV genes in order to prolong the infection cycle and/or reduce baculovirus replication and/or budding to further optimize production of recombinant proteins.

Biography

Marc Aucoin is an Associate Professor of Chemical Engineering and Academic Director of the Waterloo Engineering Professional Development Program at the University of Waterloo (Waterloo, Ontario, Canada). Prior to becoming a faculty member, he was a Technical Officer at the Biotechnology Research Institute at the National Research Council of Canada. His research interests include developing robust scalable production processes for complex biologics in animal cell culture using a combination of process and genetic strategies.

Willem "Wian" A. de Jongh, PhD
CSO, ExpreS2ion Biotechnologies
HER2 Cancer Vaccine Optimization by Combining Drosophila S2
Insect Cell Manufacturing with a Novel VLP-Display Technology

Abstract

Breast cancer is a widespread oncology indication affecting more than 1.3 million people worldwide annually, 20–30% of which are HER2 positive. HER2 is a tyrosine kinase receptor that is frequently overexpressed in several solid-tumor cancers (including breast, prostate, gastric, esophageal, and osteosarcoma) where it denotes an aggressive phenotype, high metastatic rate, and poor prognosis. In a human context, passive HER2-targeted immunotherapy using monoclonal antibodies (mAb; e.g., Trastuzumab and Pertuzumab) has proven to be an effective treatment modality which has dramatically improved clinical outcomes. Unfortunately, mAb therapy is very expensive and the repeated injections of high doses can be associated with severe side-effects that reduce efficacy.

Vaccines are highly cost-effective, but overall progress in development of anti-cancer vaccines based on cancer-associated antigens (e.g., HER2) has been hampered by inherent immune-tolerogenic mechanisms rendering the immune system incapable of reacting against the body’s own cells/proteins (i.e., self-antigens). Consequently, many attempts to develop anti-cancer vaccines have failed in clinical trials due to insufficient immunogenicity. To circumvent this central issue, we have developed a proprietary virus-like particle (VLP)-based vaccine delivery platform. Notably, the VLP platform is currently the only available technology to effectively facilitate multivalent “virus-like” display of large/complex vaccine antigens. This is key to overcome immune-tolerance and enable induction of therapeutically potent antibody responses directed against cancer-associated self-antigens.

In this talk I will discuss the non-viral Drosophila S2 insect cell production system and how it was applied to the production of HER2/neu antigen, including using advanced production methods such as perfusion for clinical material manufacture. Furthermore, I will present our data from a transgenic mouse model for spontaneous breast cancer development, where high-density display of the HER2 extracellular domain on the surface of virus-like particles (VLPs) enables induction of therapeutically potent anti-HER2 responses. Split-protein tag/catcher conjugation was used to facilitate directional covalent attachment of HER2 to the surface of icosahedral bacteriophage-derived VLPs, thereby harnessing the VLP platform to effectively overcome B-cell tolerance. Vaccine efficacy was demonstrated both in prevention and therapy of mammary carcinomas in HER2 transgenic mice. Thus, the HER2-VLP vaccine shows promise as a new strategy for treatment of HER2-positive cancer. The synergy between Drosophila S2 antigen expression and enhanced immunogenicity through VLP display also provides the opportunity to address a range of diseases where current approaches are suffering from production related or low vaccine efficacy issues.

Biography

Dr. de Jongh (South African) has a MSc in Chemical Engineering from the University of Stellenbosch, South Africa and was awarded a doctorate in Biotechnology from the Technical University of Denmark (DTU). Dr. de Jongh is CSO and co-founder of NASDAQ First North listed ExpreS2ion Biotechnologies, and CEO of AdaptVac (a joint venture co-owned by ExpreS2ion Bio). Furthermore, Dr. de Jongh was awarded an affiliated associate professorship at DTU in 2017. Dr. de Jongh has 12 years' experience in the biopharmaceutical industry.

Otto-Wilhelm Merten, PhD
Head of Applied Vectorology and Innovation, Généthon
Towards Routine Manufacturing of Gene Therapy Drugs –
Requirements for Further Improvements – Example: AAV

Abstract

With the recent marketing authorization of several gene therapy treatments based on AAV and MLV vectors, and with those to come in the near future, gene therapy has reached an almost mature state. However, in view of their definite establishment as routine therapeutic treatments, the still existing bottlenecks particularly with respect to manufacturing, quality, and potency issues are immense, at least for certain disorders, and have to be overcome. In the case of AAV vectors, the real break-through for their clinical application and finally marketing authorization was achieved after the establishment of scalable production systems. As an example, the first authorized gene therapy treatment of LPL deficiency (Glybera®) based on AAV1 vectors should be mentioned here because it is produced using the scalable baculovirus-insect cell expression system. Since LPL deficiency is an ultra-rare disorder, production scales of several tens of liters are sufficient for the production of the required doses. However, when dealing with less rare diseases or diseases for which whole body treatment are required, such as for the treatment of Duchenne muscular dystrophy (DMD) or other muscular disorders, such manufacturing scales are insufficient. For instance, based on studies in dogs, doses of 5x1013–1x1014 vg/kg are expected to be required for the treatment of a DMD patient. Such dose levels need further research dealing with the improvement of manufacturing systems, purification, vector quality, as well as vector potency, clearly indicating that this is a multi-task developmental activity.

The present talk will briefly describe the different rAAV production systems, their production capacity at this stage, as well as vector amounts required for some of the ‘famous’ indications. Since Généthon is developing the baculovirus expression system for the production of AAV vectors, several improvements, with respect to vector quantity (per production run) and quality, developed during recent years will be presented. Further improvements, in particular with respect to vector potency in a liver setting, will be briefly touched on. For perspective, the combination of all improvements will allow an improvement in manufacturing by more than fifty-fold.

Biography

Otto-Wilhelm Merten has a degree in biotechnology (PhD, 1984, University of Natural Resources and Life Sciences [BOKU] in Vienna, Austria) and today heads the Applied Vectorology and Innovation group at Généthon. He gained vast scientific experience during his time at the Institute Pasteur (Paris, France) as well as the Sandoz Research Institute (Vienna, Austria). Over many years, he has dealt with the development and optimization of serum-free and animal-free media for the cultivation of various cell lines (hybridomas, Vero, BHK 21, MDCK, etc.) and the production of different biologicals including monoclonal antibodies, recombinant proteins, and various viruses. In addition, he was involved in the development of processes for the production of viruses for vaccines (influenza, rabies, polio). During the last few years he has been involved in the development and scale-up of production and purification processes for viral vectors starting at the Institut Pasteur in Paris and continuing as head of the department of Bioprocess Development at Généthon. Since 2010 he has been in charge of the Applied Vectorology and Innovation group in view of the optimisation of vector production.

As an expert in animal cell technology, he was a member of the executive committee of the European Society for Animal Cell Technology (ESACT) for 16 years and served as its chairman from 2001 to 2005. In this context he was meeting chairman of the 15th ESACT Meeting ‘New Developments and New Applications in Animal Cell Technology’ in Tours, France in 1997. Furthermore, he has been involved in the organization of many other scientific meetings in Europe and overseas.

Since 1997 he has been editor-in-chief of Cytotechnology and served on the editorial board of Human Gene Therapy, Molecular Therapy, and Bioprocess International. Since 2012 he has been a visiting professor at ITQB/UNL, Oeiras, Portugal. His key research interests are animal cell culture, virus and viral vector production and purification and, in particular, optimisation of expression/production systems. He has profound expertise in the development and manufacturing issues of murine leukemia virus (MLV), lentivirus, adeno-associated viral vectors, as well as baculovirus.

Gary W. Blissard, PhD
Professor, Insect Cell Biology, Boyce Thompson Institute
Comparative Transcriptome Analysis of AcMNPV Infection in the
Midgut of Host Insect Trichoplusia ni, and in a T. ni Cell Line

Abstract

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is highly pathogenic to larvae of the lepidopteran insect Trichoplusia ni. Infection of the animal is initiated by occlusion derived virus (ODV) infection of the midgut epithelial cells, followed by production of budded virus (BV) and infection of other tissues within the animal. We previously performed a transcriptomic analysis of BV infection in Tnms42 cells (an alphanodavirus-free T. ni cell line derived from High Five cells). In recent studies, we used a similar transcriptomic approach to examine ODV-mediated AcMNPV infection in the T. ni midgut. We analyzed AcMNPV gene expression in infected midgut and also documented responses of midgut cells to AcMNPV infection, using a previously described AcMNPV reference transcriptome combined with a T. ni genome sequence that was generated and assembled from larval DNA. T. ni larvae were orally infected with AcMNPV occlusion bodies, and midguts were isolated for RNA sequencing over a 72 hour period. Midgut RNA-seq reads were mapped to the ~156 genes of the AcMNPV genome, and viral gene expression was analyzed and compared with prior studies of infection in a T. ni cell line. We also analyzed global transcriptional responses of midgut cells to infection by performing differential expression analysis between uninfected and infected midgut tissue.

Biography

Gary Blissard is a Professor and Virologist at the Boyce Thompson Institute at Cornell University, and an Adjunct Professor in the Departments of Microbiology and Immunology, and Entomology at Cornell University. Studies in his lab focus on virus-insect interactions with an emphasis on protein trafficking of viral envelope proteins, analyses of interactions between viral and host cell genomes at the transcriptome level, and cell line development. Gary studied polydnaviruses and obtained a PhD from Texas A&M University (with Max Summers), and studied baculovirus gene expression as a postdoc (with George Rohrmann) at Oregon State University.

Jacek Lubelski, PhD
Director Vector & Process Development, uniQure N.V.
rAAV Vector Development and Large-Scale Manufacturing Using BEVS Technology

Abstract

Recombinant adeno-associated virus (rAAV) is becoming a vector of choice for a variety of human gene therapy applications. Therefore, there is an increasing requirement for generation of a high quantity of potent rAAV vectors based on various serotypes. Baculoviruses and insect cells has been shown not only to be capable of generation of a massive quantity of high quality rAAV, but also to be a versatile system that efficiently supports rAAV vector development. I will review uniQure’s effort to use BEVs for rAAV vector development with the focus on capsids and promoters. Furthermore, in order to exploit the BEVS potential for support of large-scale rAAV manufacturing, I will present our experience with baculoviruses/insect cell system in a stirred tank bioreactor.

Biography

Jacek Lubelski, PhD joined uniQure (formerly Amsterdam Molecular Therapeutics) as a scientist in 2008. In 2017 he assumed responsibilities of Director of the Vector and Process Development Department. The Vector Development team at uniQure is responsible for molecular design, engineering, and development of viral vectors for gene therapy. The Process Development team provides non-GMP manufacturing capabilities and holds responsibility for process and manufacturing platform development. Dr. Lubelski graduated in biotechnology from Maria Curie-Skłodowska University, Lublin, Poland and received a PhD in molecular biology from the University of Groningen, The Netherlands.

Chun C. Chen, PhD
CRO, Astrid Pharma
HepE Viral Nanoparticle (HEVNP): A Multifunctional Platform for Oral Delivery

Abstract

A majority of vaccines are administered by injection that often requires cold-chain shipping and well trained personnel to administer. The World Health Organization (WHO) estimates that annually 21 million hepatitis B infections, 2 million hepatitis C infections, and 260,000 HIV/AIDS cases may be caused by re-use of syringes without sterilization. In comparison, an oral vaccine is a convenient and preferred administration route with the highest patient compliance. Like the native virus, HEVNP is stable in an acidic environment and resistant to proteolytic digestion, thus it poses a great advantage as an oral delivery vehicle. HEVNPs, with a foreign epitope exposed on the surface, can elicit mucosal and systemic immune response after oral administration. Nonetheless, HEVNP can orally deliver plasmid DNA to the epithelial cells of the small intestine and induce full immune responses against the DNA encoded antigen. Thus HEVNP can be a good carrier for oral vaccines to prevent various diseases by oral administration.

Biography

Dr. C.C. Chen is co-founder and CRO of Astrid Pharma Corp. (APC), a University of California, Davis start-up company established in June 2017. APC is taking advantage of the basic structural analysis of HepE virus-like particles in Prof. Holland Cheng’s lab at UC Davis, and proposing an oral vaccine and therapeutic delivery carrier using HepE virus-like particles. Dr. Chen obtained an MS degree in material engineering at University of Cincinnati. Then he worked 11 years in research and development in the semiconductor/LCD industry before he started his PhD research in Biotechnology at Academia Sinica, Taipei, Taiwan.

Isabelle Knott, PhD
Director, Head of GQC Biology and Raw Materials, GlaxoSmithKline Biologicals
Cervarix Vaccine: From Baculovirus Technology to the First Human BEVS-Based Vaccine
Jamal Meghrous
Senior Scientist, Protein Sciences Corporation, A Sanofi Company

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