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Expression and Purification of Virulence Factors
A Novel Approach for Identifying Drug Targets for
Autoimmune and Inflamma...
Viral Logic Systems Technology
VLST
• Privately-held company founded in 2004
• ~35 Employees
• Focused on exploiting viral...
Virulence Factors as a
Novel Route to Therapeutics
Identify
virulence
factors
Identify
cellular
targets
Define biologic
co...
Discovery of Viral TNF Receptor
Key Step in Development of Enbrel®
Smith et al (1990) Science 248: 1019
Smith et al (1991)...
Cytokines, Chemokines and Their
Receptors Encoded by Herpes Viruses
Alcami (2003)
Nat Rev
Immunol 3: 36
Genomic Scale Search
for Viral Virulence Factors
~18,500 viral proteins
in VLST database
~6,500 viral protein clusters of ...
Identification of Host Targets of
Virulence Factors
Target
identification
by LC-LTQ MS
Bind target(s) from conditioned
med...
Transient Expression of Viral Proteins
Challenge:
• Need high-throughput method to generate conditioned
media for target d...
Invitrogen’s FreeStyle™ MAX
Transfection System
• Optimized system for suspension CHO and HEK293 cells
– Media and transfe...
Effect of Cell line, Vector in Freestyle
•Same gene in different vectors and cell lines
•Similar expression levels, GS vec...
Viral Protein Expression
Target Discovery is a Numbers Game
Proteins expressed
Proteins screened
Targets identified
• Majo...
24-well Shake Plate Prescreen
• Reagent, time and effort wasted on large-scale transients, if
protein doesn’t express
• Ty...
CHO-EBNA transients, Anti HA blot
CHO Expression, 24-well Plate
High-throughput Prescreen
•~30% (21 of 70) expressed in
CH...
• “HAC”- tandem affinity tag expressed on one end of
virulence factor
• Multi-epitope tag, used for purification and targe...
Expression of N- and C-tagged
Viral Proteins
• Gene synthesize both N- and C-term. tagged versions
• >90% express at least...
Viral Protein Expression
Salvage Strategy
Express in 293’s
24-well pre-screen?
100 mL scale-up
and screen
Yes
No Express i...
• Plan on evaluating some viral factors in vivo, mouse
inflammation models
• Need quantity of protein that can’t easily be...
Clonepix-Based Colony Picking
•Secreted product detected via fluor.
labeled probe
•Evaluate fluorescence compared to
colon...
Purification Development
High-throughput Screening
Atoll GmbH
Atoll Media scout kit
•100 µL / 200 µL formats
•8 diff. resi...
HTS-Capacity Determination
•Plate format quickly identified high capacity CEX
resin
Multi-Angle Light Scattering To
Characterize Viral Proteins
UV LS RI
Wyatt TREOS
Wyatt Optilab
DSP RefractometerTosoh 3000...
5480-FLAG MALS
UV trace
Total mass (Peptide + CHO)
226 kDa
Peptide: 160 kDa
CHO: 66 kDa
5480-HAC monomer
Theor. peptide MW...
VLST Platform Highlights
•Identified numerous immunologically relevant targets
•Validated targets of 4 approved, 11 invest...
Acknowledgements
Bioinformatics and Proteomics
• Stefan Ponko, Ph.D.
• Ajamete Kaykas, Ph.D.
Protein Sciences Group
• Jeff...
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2010 Pep Talk Presentation

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2010 Pep Talk Presentation

  1. 1. Expression and Purification of Virulence Factors A Novel Approach for Identifying Drug Targets for Autoimmune and Inflammatory Diseases David Bienvenue, Ph.D. VLST Corporation
  2. 2. 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: July 8th, 2009, 3:43 PM Office view: The other 364 days of 2009
  3. 3. Virulence Factors as a Novel Route to Therapeutics Identify virulence factors Identify cellular targets Define biologic consequences of interaction Develop therapeutics mimicking virulence factors Drug Development Strategy • 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
  4. 4. Discovery of Viral TNF Receptor Key Step in Development of Enbrel® Smith et al (1990) Science 248: 1019 Smith et al (1991) BBRC 176: 335 Shope fibroma virus-T2 TNFR2 (p75) TNFR1 (p55) Fc Enbrel® (Entanercept) Cys-rich domains Dimerization domain 29 % sequence identity 38 % sequence identity
  5. 5. Cytokines, Chemokines and Their Receptors Encoded by Herpes Viruses Alcami (2003) Nat Rev Immunol 3: 36
  6. 6. Genomic Scale Search for Viral Virulence Factors ~18,500 viral proteins in VLST database ~6,500 viral protein clusters of similar proteins >600 putative virulence factors identified and queued for screening Bioinformatic Expert System selection criteria Topology – anchor, secreted, other transmembrane Homology to human proteins Species Sequence cluster membership N-Linked glycosylation sites Virulence Pfam motifs Non-essential for viral replication Analyzed 267 viral genomes 98 pox viruses 96 herpes viruses 62 adeno viruses 11 asfar viruses
  7. 7. Identification of Host Targets of Virulence Factors Target identification by LC-LTQ MS Bind target(s) from conditioned media, cell lysates from immune-related cell lines, using tandem affinity tag Transiently express tagged viral proteins Bioinformatic mining for virulence factors Synthesize viral genes VF VF VF FACS screen and Bioassay panel
  8. 8. Transient Expression of Viral Proteins Challenge: • 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
  9. 9. 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
  10. 10. Effect of Cell line, Vector in Freestyle •Same gene in different vectors and cell lines •Similar expression levels, GS vector has slight advantage Comparison of Day 4 Titers 40.9 56.3 36.8 43.1 43.6 30.7 40.6 40.0 45.2 26.7 51.9 50.5 0 10 20 30 40 50 60293F-CHEF 293F-GS 293F-409 CHOF-CHEF CHOF-GS CHOF-409 CHO-EBCHEF CHO-EBGS CHO-EB-409 293-EB-CHEF 293-EB-GS 293-EB-409 ug/mL
  11. 11. Viral Protein Expression Target Discovery is a Numbers Game Proteins expressed Proteins screened Targets 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
  12. 12. 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 CHO Goal • 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
  13. 13. CHO-EBNA transients, Anti HA blot CHO Expression, 24-well Plate High-throughput Prescreen •~30% (21 of 70) expressed in CHO •Pre-screen all viral ORFs in 293 and CHO •Only perform larger transients on vectors positive for expression
  14. 14. • “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 N- And C-Tagged Virulence Factors HAC Tag Affinity Resin
  15. 15. 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
  16. 16. Viral Protein Expression Salvage Strategy Express in 293’s 24-well pre-screen? 100 mL scale-up and screen Yes No Express in CHO 24-well prescreen? Yes No STOP START 100 mL scale-up and screen •Use DNA from CRO to perform prescreen •Minimize resources for DNA prep and large scale transients “No viral protein left behind”
  17. 17. • 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 Generation of Stable CHO Cell Lines Large-scale Viral Protein Production
  18. 18. 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)
  19. 19. Purification Development High-throughput Screening Atoll GmbH Atoll Media scout kit •100 µL / 200 µL formats •8 diff. resins per class ProA Anion Cation CHA HIC Millipore Vacuum Manifold •Screen multiple conditions quickly and easily •Facilitates use of “Design of Experiments” •Consumes little protein
  20. 20. HTS-Capacity Determination •Plate format quickly identified high capacity CEX resin
  21. 21. Multi-Angle Light Scattering To Characterize Viral Proteins UV LS RI Wyatt TREOS Wyatt Optilab DSP RefractometerTosoh 3000SWxl •Determine multimer state of novel viral proteins •Often have several glycosylation sites •Shape-dependent effects increase error of MW derived from std. curve of other proteins Solution: Use SEC with Multi-Angle Light Scattering Agilent HPLC
  22. 22. 5480-FLAG MALS UV trace Total mass (Peptide + CHO) 226 kDa Peptide: 160 kDa CHO: 66 kDa 5480-HAC monomer Theor. peptide MW: 24 kDa 160/24= ~6.7 (Multi-Angle Light Scattering)
  23. 23. VLST Platform Highlights •Identified numerous immunologically relevant targets •Validated targets of 4 approved, 11 investigational drugs •Partnership with Novo Nordisk in 2008 to provide research targets •Therapeutic programs based on viral targets entering the clinic in 2010/2011
  24. 24. Acknowledgements Bioinformatics 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

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