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2010 Pep Talk Presentation






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    2010 Pep Talk Presentation 2010 Pep Talk Presentation Presentation Transcript

    • Expression and Purification of Virulence Factors A Novel Approach for Identifying Drug Targets for Autoimmune and Inflammatory Diseases David Bienvenue, Ph.D. VLST Corporation
    • Viral Logic Systems Technology VLST• Privately-held company founded in 2004• ~35 Employees• Focused on exploiting viral evolution to develop novel biotherapeutics• Based in “sunny” Seattle, WA Office view: Office view: July 8th, 2009, 3:43 PM The other 364 days of 2009
    • Virulence Factors as a Novel Route to Therapeutics• Some viral proteins modulate/suppress host immune system• Facilitate viral infection and influence severity of disease• Can be homologous or unrelated to host genes• Targets of viral proteins validated as treatment methods for autoimmune/inflammatory illness Drug Development Strategy Identify Identify Define biologic Develop therapeuticsvirulence cellular consequences mimicking factors targets of interaction virulence factors
    • Discovery of Viral TNF Receptor Key Step in Development of Enbrel®TNFR1 Shope fibroma TNFR2 Enbrel® (p55) virus-T2 (p75) (Entanercept) 29 % sequence 38 % sequence identity identity Cys-rich domains Dimerization domain Fc Smith et al (1990) Science 248: 1019 Smith et al (1991) BBRC 176: 335
    • Cytokines, Chemokines and Their Receptors Encoded by Herpes VirusesAlcami (2003) Nat RevImmunol 3: 36
    • Genomic Scale Search for Viral Virulence Factors Analyzed 267 viral genomes 98 pox viruses ~18,500 viral proteins 96 herpes viruses in VLST database 62 adeno viruses 11 asfar viruses Bioinformatic Expert System~6,500 viral protein clusters of similar selection criteria proteins Topology – anchor, secreted, other transmembrane Homology to human proteins Species Sequence cluster membership >600 putative virulence factors N-Linked glycosylation sites identified and queued for screening Virulence Pfam motifs Non-essential for viral replication
    • Identification of Host Targets of Virulence FactorsBioinformatic mining for Synthesize Transiently express virulence factors viral genes tagged viral proteins VF VF FACS screen and Target Bioassay panel identification by LC-LTQ MS VF Bind target(s) from conditioned media, cell lysates from immune-related cell lines, using tandem affinity tag
    • Transient Expression of Viral ProteinsChallenge:• Need high-throughput method to generate conditioned media for target discovery (goal ~20-30/wk)• Adherent transfections not easily scaleable• Scale up via technology, not FTE’s!Solution:• Transition away from adherent 293-EBNA’s, to suspension transient transfections
    • Invitrogen’s FreeStyle™ MAX Transfection System• Optimized system for suspension CHO and HEK293 cells – Media and transfection reagent chemically defined, serum free – Cells adapted for suspension culture in FreeStyle™ media• One shake flask vs. many T-flasks – Less labor intensive, higher throughput
    • Effect of Cell line, Vector in Freestyle Comparison of Day 4 Titers 60 56.3 51.9 50.5 50 43.1 43.6 45.2 40.9 40.6 40.0 40 36.8 ug/mL 30.7 30 26.7 20 10 0 F- GS F GS 9 S F 409 EF - 409 - GS 09 HEF F- 40 CHE - CHE EB- G EB- 4 F- CH -EB -EB- 293F 293F EB- C CHO CHO -EB 293F 293- CHO 293- CHO CHO 293- CHO •Same gene in different vectors and cell lines •Similar expression levels, GS vector has slight advantage
    • Viral Protein ExpressionTarget Discovery is a Numbers GameProteins expressedProteins screenedTargets identified• Majority of putative virulence factors express in 293’s• Some 293 non-expressors can be expressed in CHO• Maximize number of viral factors going into screen• Minimize effort, reagents spent on non-expressors
    • 24-well Shake Plate Prescreen• Reagent, time and effort wasted on large-scale transients, if protein doesn’t express• Typically if vectors failed in 293, re-try in CHOGoal• Minimize effort re-transfecting viral proteins in CHO• Attempt prescreen expression in in 24-well shaker plates – Mimic conditions in 100 mL shake flasks – Try ~70 constructs that didn’t previously express in 293’s
    • CHO Expression, 24-well Plate High-throughput Prescreen •~30% (21 of 70) expressed in CHO •Pre-screen all viral ORFs in 293 and CHOCHO-EBNA transients, Anti HA blot •Only perform larger transients on vectors positive for expression
    • N- And C-Tagged Virulence Factors Affinity Resin HAC Tag• “HAC”- tandem affinity tag expressed on one end of virulence factor• Multi-epitope tag, used for purification and target discovery• Possibility that HAC may block binding to target• May also impact protein expression/secretion
    • Expression of N- and C-tagged Viral Proteins• Gene synthesize both N- and C-term. tagged versions• >90% express at least one version – ~20% increase over expressing C-tag alone• Increases probability of identifying targets – In some cases, only one version binds target
    • Viral Protein Expression Salvage Strategy START Express in 293’s No Express in CHO 24-well 24-well pre-screen? prescreen? Yes No Yes 100 mL scale-up and screen 100 mL scale-up and screen STOP•Use DNA from CRO to perform prescreen•Minimize resources for DNA prep and large scale transients “No viral protein left behind”
    • Generation of Stable CHO Cell LinesLarge-scale Viral Protein Production• Plan on evaluating some viral factors in vivo, mouse inflammation models• Need quantity of protein that can’t easily be obtained via transient transfection• Codon-optimize gene expression in mammalian cells• Add restriction sites to subclone into CHEF (CMC- ICOS) or GS (Lonza) expression vectors• Utilize Genetix “Clonepix” colony picker to facilitate clone picking process
    • Clonepix-Based Colony Picking •Secreted product detected via fluor. labeled probe •Evaluate fluorescence compared to colony size •Useful method for weeding out low/non- expressing clones •Permits screening thousands, vs. hundreds of colonies •Results in increased titer of final production clone (relative to limiting dilution cloning and picking)
    • Purification DevelopmentHigh-throughput Screening Atoll Media scout kit •100 µL / 200 µL formats •8 diff. resins per class ProA Anion Atoll GmbH Cation CHA HIC Millipore Vacuum Manifold •Screen multiple conditions quickly and easily •Facilitates use of “Design of Experiments” •Consumes little protein
    • HTS-Capacity Determination•Plate format quickly identified high capacity CEXresin
    • Multi-Angle Light Scattering To Characterize Viral Proteins Agilent HPLC UV LS RI Wyatt Optilab Tosoh 3000SWxl Wyatt TREOS DSP Refractometer•Determine multimer state of novel viral proteins•Often have several glycosylation sites•Shape-dependent effects increase error of MW derivedfrom std. curve of other proteinsSolution: Use SEC with Multi-Angle Light Scattering
    • 5480-FLAG MALS(Multi-Angle Light Scattering) 5480-HAC monomer Total mass (Peptide + CHO) Theor. peptide MW: 24 kDa 226 kDa 160/24= ~6.7 Peptide: 160 kDa UV trace CHO: 66 kDa
    • VLST Platform Highlights•Identified numerous immunologically relevant targets •Validated targets of 4 approved, 11 investigational drugs•Partnership with Novo Nordisk in 2008 to provideresearch targets•Therapeutic programs based on viral targets entering theclinic in 2010/2011
    • AcknowledgementsBioinformatics and Proteomics• Stefan Ponko, Ph.D.• Ajamete Kaykas, Ph.D.Protein Sciences Group• Jeff Bartron• Chris Tompkins• Laura Hajny• Patrick Mosher• Ryan Kelly• Ryan Merrill