tapboost pegs boston slide ver 2015 06-23

TAPBOOSTTAPBOOST®®

Breakthrough Platform to Improve Protein Production

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


Protein Folding System and Quality Control Protein Folding System and Quality Control SystemSystem

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


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







Golgi

ER

TAPBOOSTTAPBOOST®® ((TaTargeted rgeted PProtein rotein BoostBoosting Platform) ing Platform) Discovery-2Discovery-2

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


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


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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®®

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®®



specifically enhances the secretion of the target proteinsTAPBOOSTTAPBOOST®®

B

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)


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


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


anti-TNF Ab(Humira)

anti-VEGF Ab(Avastin)

anti-IL8 Ab(DP12)

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Fol

d In

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se

Avastin

Fol

d In

crea

se

Fol

d In

crea

se

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

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

Fo

ld in

crea

se

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


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Fol

d in

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se

TNFRI-Fc (IgG1)

TNFα Binding Ability (ELISA)

TNFRI-Fc (IgG4)

Platform: Fc Fusion of hIgG4Platform: Fc Fusion of hIgG4TAPBOOSTTAPBOOST®®


enhances the production of both types of Fc fusion proteinTAPBOOSTTAPBOOST®®

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®®


Protein Aggregation Experiment conducted by Major Pharma Partner in Pilot Project

• PD group wanted to know whether can also results in lower aggregation.

CharacterizationCharacterization


reduces the aggregation of produced proteinsTAPBOOSTTAPBOOST®®


Target Protein(Conditioned Media)

Fol

d In

crea

se (

%)

IL13Rα2-Fc

IL13Rα2-Fc

Protein Production in CHO Suspension conducted by CRO

050

100150

200250

300350

400450

enhances the production in CHO Suspension Expression System

Transition from 293T to CHO suspension Transition from 293T to CHO suspension systemsystem





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®®



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



Application for Microbial ExpressionApplication for Microbial Expression



increases both expression and solubility of the target protein in bacteriaTAPBOOSTTAPBOOST®®

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®®


• Any kind of protein can be expressed by platform• Easy implementation into your current bioprocess• Combine with any other technology for synergistic effect



tapboost pegs boston slide ver 2015 06-23

Technology

protein binding sequence

protein production

sequence effecter sequence

production of target

combinations targeted

model protein il

domain fc binding sequence

intended target protein