Download - Tapboost pegs boston slide ver 2015 06-23
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TAPBOOSTTAPBOOST®®
Breakthrough Platform to Improve Protein Production
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Misfolded Protein
Protein/Ab StabilityProtein/Ab SecretionFunctional ProteinImmunogenicity
human cell
Polypeptide
Protein Folding
· Suppression of Translation
· Induction/Expression of Chaperones
· Cell suicide
Platform enhances Protein Production of the Target Proteins by controlling Protein Folding/Cell Quality Control System
RNA
gene
Folded Protein
· Degradation of proteins Degradation
RNA
Protein Folding
Cell Quality Control System
human cell
Total yield of Biologics/Ab
TAPBOOSTTAPBOOST®®
Protein Folding System and Quality Control Protein Folding System and Quality Control SystemSystem
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Screening System
Step 1: in silico Screening for Protein Domain/Subdomain
Misfolding-Prone Protein
DegradationPoor Secretion
Protein Stability Protein Secretion
Step 3Protein Stability and Secretion Test
Develop the technology which controls protein folding/cellular quality control system specifically for an intended target protein
GOAL
Through the screening, we discovered the amino acid sequence with protein stability and secretion activity
First Year
Step 2: Library Construction
((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) DiscoveryDiscovery
TAPBOOSTTAPBOOST®®
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Development of Platform
Step 4: Construction of
Targeting domain (bind to a target protein)
Discovered sequence(Effecter Sequence)
Targeting Domain• Fc binding sequence: 14 combinations• Antibody binding sequence: 5 combinations• Target specific sequence: 9 combinations• Unfolding protein binding sequence: 5 combinations
Flexible Linker / Rigid Liner• 5 combinations
Effecter Sequence
Signal Sequence• 4 combinations
Targeted Protein
Second Year
Step 5: Enhancement of the Production of Target Protein by
PlatforPlatformm
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
Golgi
ER
TAPBOOSTTAPBOOST®® ((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) Discovery-2Discovery-2
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Discovered sequence
Sequence Optimization
Deletion ofthe sequence(49 deletion mutants)
Minimum sequence(9 amino acid sequence)
Step 6: Identification of the Minimum Sequence
Various mutants of the sequence(49 mutations)
Step 7: Formulation of the Sequence
X1 X2 X3 X4 X5 X6 X7 X8 X9
Formulation of the Amino Acid Sequence
((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) Discovery-3Discovery-3
TAPBOOSTTAPBOOST®®
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Model Protein; IL-13 receptor 2 (IL13R2)-Fc fusion protein
CHALLENGE: The expression levels of IL13R2 are poor, and the protein is prone to misfolding and aggregation.
Cell
IL13R2-Fc
ERNu
• Disease Relevance: a key mediator of allergic inflammation
Aggregation of IL13R2-Fc
Only properly folded IL13R2-Fc secrets
• Fc fusion protein with sIL13R2 (sIL13R2-Fc) has been developed for the treatment of asthma
• Production of sIL13R2-Fc using human cells is inefficient due to its instability
• Soluble IL13R2 (sIL13R2) blocks the signal from IL-13 as a decoy receptor
Application: IL13RApplication: IL13Rαα2-Fc Protein Case 2-Fc Protein Case StudyStudy
TAPBOOSTTAPBOOST®®
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SecretedIL13R2-Fc
IL13R2-FcInside Cells
Secreted IL13R2-Fc IL13R2-Fc Inside Cells
IL13R2-Fc
Fol
d In
crea
se (
%)
Fol
d In
crea
se (
%)
IL13R2-Fc
Case Study - IL13RCase Study - IL13Rαα2-Fc 2-Fc ProteinProteinTAPBOOSTTAPBOOST®®
enhances the secretion of IL13Rα2-Fc ProteinTAPBOOSTTAPBOOST®®
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120>20x
TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
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ER
Targeted ProteinTargeting Domain was designed to bind only to Protein B
SecretedTarget Protein
(A or B)
A A A B B B B BTarget Protein
Platform: Target Protein SpecificityPlatform: Target Protein SpecificityTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
specifically enhances the secretion of the target proteinsTAPBOOSTTAPBOOST®®
B
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enhances the production of all three tested antibodies
Antibody Production Detection: Western Blot Assay using anti-hIgG antibody
Platform: Antibody ExamplesPlatform: Antibody ExamplesTAPBOOSTTAPBOOST®®
Western(anti-hIgG Ab)
TAPBOOSTTAPBOOST®®
Target Antibody
anti-IL8 Ab(DP12)
anti-TNF Ab(Humira)
anti-VEGF Ab(Avastin)
antibody Brand NameProduction Increase
(produced protein)
anti-IL8 Ab DP12 6.4 +/- 0.89
anti-TNFα Ab Humira 6.3 +/- 0.33
anti-VEGF Ab Avastin 3.5 +/- 0.27
TAPBOOSTTAPBOOST®®
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Antibody binding affinity for its corresponding epitopeDetection: ELISA using rVEGF, rTNFα, or rIL-8 for Avastin, Humira, or DP12, respectively
Platform: Antibody ExamplesPlatform: Antibody ExamplesTAPBOOSTTAPBOOST®®
Antibodies produced by hold activity
TAPBOOSTTAPBOOST®®
anti-TNF Ab(Humira)
anti-VEGF Ab(Avastin)
anti-IL8 Ab(DP12)
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Avastin
Fol
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Fol
d In
crea
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Humira DP12
TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
antibody Brand NameProduction Increase
(produced protein)
Production Increase
by ELISA
anti-VEGF Ab Avastin 3.5 +/- 0.27 4.2 +/- 0.19
anti-TNFα Ab Humira 6.3 +/- 0.33 6.0 +/- 0.82
anti-IL8 Ab DP12 6.4 +/- 0.89 7.3 +/- 0.56
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substantially enhances the production of Factor VIII-Fc
Platform: Enzyme ExamplePlatform: Enzyme ExampleTAPBOOSTTAPBOOST®®
Fo
ld in
crea
se
FVIII Production
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FVIII-Fc
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ld in
crea
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FVIII Activity
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FVIII-Fc
Target ProteinProduction Increase
(produced protein)
Production Increase
by Activity
FVIII-Fc 19.2 +/- 0.47 21.3 +/- 0.23
TAPBOOSTTAPBOOST®®
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TAPBOOSTTAPBOOST®®
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d in
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TNFRI-Fc (IgG1)
TNFα Binding Ability (ELISA)
TNFRI-Fc (IgG4)
Platform: Fc Fusion of hIgG4Platform: Fc Fusion of hIgG4TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
enhances the production of both types of Fc fusion proteinTAPBOOSTTAPBOOST®®
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IL13R2-FcTNFRI-Fc1AT-Fc 1ATMT-Fc
Original Secretion of Targeted Protein
Effect of
MaximumEffect
may “rescue” problem proteins from misfolding and maximize secretion levels
works on difficult proteinsworks on difficult proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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Protein Aggregation Experiment conducted by Major Pharma Partner in Pilot Project
• PD group wanted to know whether can also results in lower aggregation.
CharacterizationCharacterization
TAPBOOSTTAPBOOST®®
reduces the aggregation of produced proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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Target Protein(Conditioned Media)
Fol
d In
crea
se (
%)
IL13Rα2-Fc
IL13Rα2-Fc
Protein Production in CHO Suspension conducted by CRO
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300350
400450
enhances the production in CHO Suspension Expression System
Transition from 293T to CHO suspension Transition from 293T to CHO suspension systemsystem
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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PK
C A
ctiv
ity (
OD
450
-BG
)
AT1 receptor-AT1 receptor
Angiotensin II (100nM) 5min 10min 15min 30min
Angiotensin II receptor type 1(AT1 receptor)
AT1 receptor
-AT1 receptor
+ Angiontensin II
Application for Screening R&D – GPCR proteins (POC)Application for Screening R&D – GPCR proteins (POC)
enhances the expression of difficult-to-express proteins such as GPCR proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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Soluble protein (SP)
Inclusion Body (IB)
bacteria
Bacterial Genome
plasmid
We customized platform for bacterial expression system by using a proprietary algorithms and intensive search for specific amino acid sequences involved in protein folding.
Platform
Test Protein A
Test Protein B
Test Protein C
SP IBTotal SP IBTotal
SP IBTotal SP IBTotal
SP IBTotal SP IBTotal
Application for Microbial ExpressionApplication for Microbial Expression
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
increases both expression and solubility of the target protein in bacteriaTAPBOOSTTAPBOOST®®
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Advantages of
SummarySummary
Increased Productivity byTarget proteins
TNFRI-Fc (Enbrel) 480%
Factor IX-Fc 320%
Factor FVII-Fc 1000%
IL13R2-Fc protein 2000%
Factor VIII-Fc 2100%
Humira
Avastin
DP12
600%420%
730%
293T monolayer System
293T Suspension System
IL13R2-Fc protein 1100%CHO Suspension System
IL13R2-Fc protein 400%* Optimization process is in progress
*
*
(%)TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
• Any kind of protein can be expressed by platform• Easy implementation into your current bioprocess• Combine with any other technology for synergistic effect
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®