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TAPBOOSTTAPBOOST®®
Breakthrough Platform to Improve Protein Production
Misfolded Protein
Protein/Ab Stability
Protein/Ab Secretion
Functional Protein
Immunogenicity
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 ControlProtein Folding System and Quality Control
SystemSystem
Screening System
Step 1: in silico Screening for Protein Domain/Subdomain
Misfolding-Prone
Protein
Degradation
Poor Secretion
Protein Stability
Protein Secretion
Step 3
Protein 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
((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform)
DiscoveryDiscovery
TAPBOOSTTAPBOOST®®
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
PlatforPlatfor
mm
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
Golgi
ER
TAPBOOSTTAPBOOST®® ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform)
Discovery-2Discovery-2
Discovered sequence
Sequence Optimization
Deletion of
the 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
((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform)
Discovery-3Discovery-3
TAPBOOSTTAPBOOST®®
Model Protein; IL-13 receptor α2 (IL13Rα2)-Fc fusion protein
CHALLENGE: The expression levels of
IL13Rα2 are poor, and the protein is
prone to misfolding and aggregation.
Cell
IL13Rα2-Fc
ERNu
• Disease Relevance: a key mediator of allergic inflammation
Aggregation of
IL13Rα2-Fc
Only properly folded
IL13Rα2-Fc secrets
• Fc fusion protein with sIL13Rα2 (sIL13Rα2-Fc) has been developed for the
treatment of asthma
• Production of sIL13Rα2-Fc using human cells is inefficient due to its
instability
• Soluble IL13Rα2 (sIL13Rα2) blocks the signal from IL-13 as a decoy
receptor
Application: IL13RApplication: IL13Rαα2-Fc Protein Case2-Fc Protein Case
StudyStudy
TAPBOOSTTAPBOOST®®
0
500
1000
1500
2000
2500
0
20
40
60
80
100
120
Secreted
IL13Rα2-Fc
IL13Rα2-Fc
Inside Cells
Secreted IL13Rα2-Fc IL13Rα2-Fc Inside Cells
IL13Rα2-Fc
FoldIncrease(%)
FoldIncrease(%)
IL13Rα2-Fc
Case Study - IL13RCase Study - IL13Rαα2-Fc2-Fc
ProteinProtein
TAPBOOSTTAPBOOST®®
enhances the secretion
of IL13Rα2-Fc Protein
TAPBOOSTTAPBOOST®®
0
500
1000
1500
2000
2500
0
20
40
60
80
100
120
>20x
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
ER
Targeted Protein
Targeting Domain was designed
to bind only to Protein B
Secreted
Target 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 proteins
TAPBOOSTTAPBOOST®®
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)
TAPBOOSTTAPBOOST®®
Target Antibody
anti-IL8 Ab
(DP12)
anti-TNFα Ab
(Humira)
anti-VEGF Ab
(Avastin)
antibody Brand Name
Production
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®®
Antibody binding affinity for its corresponding epitope
Detection: 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-TNFα Ab
(Humira)
anti-VEGF Ab
(Avastin)
anti-IL8 Ab
(DP12)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
FoldIncrease
Avastin
FoldIncrease
FoldIncrease
Humira DP12
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
antibody Brand Name
Production
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®®
Foldincrease
FVIII Production
0
2
4
6
8
10
12
14
16
18
20
FVIII-Fc
Foldincrease
FVIII Activity
0
2
4
6
8
10
12
14
16
18
20
22
24
FVIII-Fc
Target Protein
Production Increase
(produced protein)
Production Increase
by Activity
FVIII-Fc 19.2 +/- 0.47 21.3 +/- 0.23
TAPBOOSTTAPBOOST®®
0
2
4
6
8
10
12
14
16
18
20
TAPBOOSTTAPBOOST®®
0
2
4
6
8
10
12
14
16
18
20
22
24
TAPBOOSTTAPBOOST®®
0
2
4
6
8
10
12
Foldincrease
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 protein
TAPBOOSTTAPBOOST®®
IL13Rα2-FcTNFRI-Fcα1AT-Fc α1ATMT-Fc
Original Secretion of
Targeted Protein
Effect of
Maximum
Effect
may “rescue” problem proteins from
misfolding and maximize secretion levels
works on difficult proteinsworks on difficult proteinsTAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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
TAPBOOSTTAPBOOST®®
reduces the aggregation
of produced proteins
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
Target Protein
(Conditioned Media)
FoldIncrease(%)
IL13Rα2-Fc
IL13Rα2-Fc
Protein Production in CHO Suspension conducted by CRO
0
50
100
150
200
250
300
350
400
450
enhances the production in CHO
Suspension Expression System
Transition from 293T to CHO suspensionTransition from 293T to CHO suspension
systemsystem
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
PKCActivity(OD450-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 proteins
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
TAPBOOSTTAPBOOST®®
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 bacteria
TAPBOOSTTAPBOOST®®
Advantages of
SummarySummary
Increased Productivity byTarget proteins
TNFRI-Fc (Enbrel) 480%
Factor IX-Fc 320%
Factor FVII-Fc 1000%
IL13Rα2-Fc protein 2000%
Factor VIII-Fc 2100%
Humira
Avastin
DP12
600%
420%
730%
293T monolayer System
293T Suspension System
IL13Rα2-Fc protein 1100%
CHO Suspension System
IL13Rα2-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®®

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PEGS Boston slide strategia

  • 2. Misfolded Protein Protein/Ab Stability Protein/Ab Secretion Functional Protein Immunogenicity 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 ControlProtein Folding System and Quality Control SystemSystem
  • 3. Screening System Step 1: in silico Screening for Protein Domain/Subdomain Misfolding-Prone Protein Degradation Poor Secretion Protein Stability Protein Secretion Step 3 Protein 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 ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform) DiscoveryDiscovery TAPBOOSTTAPBOOST®®
  • 4. 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 PlatforPlatfor mm TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® Golgi ER TAPBOOSTTAPBOOST®® ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform) Discovery-2Discovery-2
  • 5. Discovered sequence Sequence Optimization Deletion of the 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 ((TaTargetedrgeted PProteinrotein BoostBoosting Platform)ing Platform) Discovery-3Discovery-3 TAPBOOSTTAPBOOST®®
  • 6. Model Protein; IL-13 receptor α2 (IL13Rα2)-Fc fusion protein CHALLENGE: The expression levels of IL13Rα2 are poor, and the protein is prone to misfolding and aggregation. Cell IL13Rα2-Fc ERNu • Disease Relevance: a key mediator of allergic inflammation Aggregation of IL13Rα2-Fc Only properly folded IL13Rα2-Fc secrets • Fc fusion protein with sIL13Rα2 (sIL13Rα2-Fc) has been developed for the treatment of asthma • Production of sIL13Rα2-Fc using human cells is inefficient due to its instability • Soluble IL13Rα2 (sIL13Rα2) blocks the signal from IL-13 as a decoy receptor Application: IL13RApplication: IL13Rαα2-Fc Protein Case2-Fc Protein Case StudyStudy TAPBOOSTTAPBOOST®®
  • 7. 0 500 1000 1500 2000 2500 0 20 40 60 80 100 120 Secreted IL13Rα2-Fc IL13Rα2-Fc Inside Cells Secreted IL13Rα2-Fc IL13Rα2-Fc Inside Cells IL13Rα2-Fc FoldIncrease(%) FoldIncrease(%) IL13Rα2-Fc Case Study - IL13RCase Study - IL13Rαα2-Fc2-Fc ProteinProtein TAPBOOSTTAPBOOST®® enhances the secretion of IL13Rα2-Fc Protein TAPBOOSTTAPBOOST®® 0 500 1000 1500 2000 2500 0 20 40 60 80 100 120 >20x TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
  • 8. ER Targeted Protein Targeting Domain was designed to bind only to Protein B Secreted Target 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 proteins TAPBOOSTTAPBOOST®® B
  • 9. 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-TNFα Ab (Humira) anti-VEGF Ab (Avastin) antibody Brand Name Production 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®®
  • 10. Antibody binding affinity for its corresponding epitope Detection: 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-TNFα Ab (Humira) anti-VEGF Ab (Avastin) anti-IL8 Ab (DP12) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 FoldIncrease Avastin FoldIncrease FoldIncrease Humira DP12 TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® antibody Brand Name Production 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
  • 11. substantially enhances the production of Factor VIII-Fc Platform: Enzyme ExamplePlatform: Enzyme ExampleTAPBOOSTTAPBOOST®® Foldincrease FVIII Production 0 2 4 6 8 10 12 14 16 18 20 FVIII-Fc Foldincrease FVIII Activity 0 2 4 6 8 10 12 14 16 18 20 22 24 FVIII-Fc Target Protein Production Increase (produced protein) Production Increase by Activity FVIII-Fc 19.2 +/- 0.47 21.3 +/- 0.23 TAPBOOSTTAPBOOST®® 0 2 4 6 8 10 12 14 16 18 20 TAPBOOSTTAPBOOST®® 0 2 4 6 8 10 12 14 16 18 20 22 24 TAPBOOSTTAPBOOST®®
  • 12. 0 2 4 6 8 10 12 Foldincrease 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 protein TAPBOOSTTAPBOOST®®
  • 13. IL13Rα2-FcTNFRI-Fcα1AT-Fc α1ATMT-Fc Original Secretion of Targeted Protein Effect of Maximum Effect may “rescue” problem proteins from misfolding and maximize secretion levels works on difficult proteinsworks on difficult proteinsTAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
  • 14. 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 proteins TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
  • 15. Target Protein (Conditioned Media) FoldIncrease(%) IL13Rα2-Fc IL13Rα2-Fc Protein Production in CHO Suspension conducted by CRO 0 50 100 150 200 250 300 350 400 450 enhances the production in CHO Suspension Expression System Transition from 293T to CHO suspensionTransition from 293T to CHO suspension systemsystem TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
  • 16. PKCActivity(OD450-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 proteins TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®® TAPBOOSTTAPBOOST®®
  • 17. 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 bacteria TAPBOOSTTAPBOOST®®
  • 18. Advantages of SummarySummary Increased Productivity byTarget proteins TNFRI-Fc (Enbrel) 480% Factor IX-Fc 320% Factor FVII-Fc 1000% IL13Rα2-Fc protein 2000% Factor VIII-Fc 2100% Humira Avastin DP12 600% 420% 730% 293T monolayer System 293T Suspension System IL13Rα2-Fc protein 1100% CHO Suspension System IL13Rα2-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®®

Editor's Notes

  1. Conceptual background on the challenge/opportunity of protein folding in recombinant protein expression systems. TAPBOOST’s solution to these challenges.
  2. Our efforts at Boston strategics have focused on development of a value-added system using proprietary chaperone technology to optimize/improve folding.
  3. In order to create a system that produces “clean” and natural untagged sequence we improved upon the original designs. This is now protected by global patents.
  4. Conclusion: not only larger yields, but less aggregation: larger proportion of monomeric target. The other Steps, transitions to CHO, and scale-up feasibilities are moving along with other partners.
  5. GPCRs are a major class of therapeutically relevant molecules, representing more than 50% of all screening targets. Here we tested another application for the TapBoost platform- the use in producing difficult to express drug screening targets such as GPCRs.
  6. Conclusion: not only larger yields, but less aggregation: larger proportion of monomeric target. The other Steps, transitions to CHO, and scale-up feasibilities are moving along with other partners.