TAPBOOST. Breakthrough Platform to Improve Protein Production

Transcription

1 Breakthrough Platform to Improve Protein Production

2 Protein Folding System and Quality Control System Protein Folding RNA Polypeptide Misfolded Protein Cell Quality Control System Degradation of proteins Degradation gene Suppression of Translation RNA Induction/Expression of Chaperones Protein Folding Folded Protein human cell Cell suicide human cell Protein/Ab Stability Protein/Ab Secretion Functional Protein Immunogenicity Total yield of Biologics/Ab Platform enhances Protein Production of the Target Proteins by controlling Protein Folding/Cell Quality Control System

3 (Targeted Protein Boosting Platform) Discovery GOAL Develop the technology which controls protein folding/cellular quality control system specifically for an intended target protein Screening System Step 1: in silico Screening for Protein Domain/Subdomain Step 2: Library Construction First Year Step 3 Protein Stability and Secretion Test Misfolding-Prone Protein Protein Stability Protein Secretion Degradation Poor Secretion Through the screening, we discovered the amino acid sequence with protein stability and secretion activity

4 (Targeted Protein Boosting Platform) Discovery-2 Development of Platform Step 4: Construction of Discovered sequence (Effecter Sequence) Targeting domain (bind to a target protein) Step 5: Enhancement of the Production of Target Protein by Targeted Protein Second Year ER Golgi Targeting Domain Flexible Linker / Rigid Liner Effecter Sequence Signal Sequence Platform

5 (Targeted Protein Boosting Platform) Discovery-3 Step 6: Identification of the Minimum Sequence Discovered sequence Deletion of the sequence (49 deletion mutants) Step 7: Formulation of the Sequence Minimum sequence (9 amino acid sequence) Various mutants of the sequence (49 mutations) Formulation of the Amino Acid Sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9

6 Application: IL13Rα2-Fc Protein Case Study Model Protein; IL-13 receptor a2 (IL13Ra2)-Fc fusion protein a receptor a a treatment of asthma 2-Fc using human cells is inefficient due to its IL13Ra2-Fc Only properly folded IL13Ra2-Fc secrets Nu ER Cell CHALLENGE: The expression levels of IL13Ra2 are poor, and the protein is prone to misfolding and aggregation. Aggregation of IL13Ra2-Fc

7 Case Study - IL13Rα2-Fc Protein Secreted IL13Ra2-Fc IL13Ra2-Fc Inside Cells Secreted IL13Ra2-Fc Fold Increase (%) IL13Ra2-Fc >20x IL13Ra2-Fc Inside Cells Fold Increase (%) IL13Ra2-Fc enhances the secretion of IL13Rα2-Fc Protein

8 Platform: Target Protein Specificity Targeted Protein Targeting Domain was designed to bind only to Protein B ER B Target Protein A A A B B B B B Secreted Target Protein (A or B) specifically enhances the secretion of the target proteins

9 Platform: Antibody Examples Target Antibody anti-il8 Ab (DP12) anti-tnfa Ab (Humira) anti-vegf Ab (Avastin) Western (anti-higg Ab) Brand Name Production Increase (produced protein) 6.4 +/ α Humira 6.3 +/ Avastin 3.5 +/ enhances the production of all three tested antibodies

10 Platform: Antibody Examples α Fold Increase Avastin anti-vegf Ab (Avastin) Fold Increase Humira anti-tnfa Ab (Humira) Fold Increase DP12 anti-il8 Ab (DP12) Brand Name Production Increase (produced protein) Production Increase by ELISA Avastin 4.2 +/ α Humira 6.0 +/ / Antibodies produced by hold activity

11 Platform: Enzyme Example Fold increase FVIII-Fc FVIII Production Fold increase FVIII-Fc FVIII Activity Target Protein Production Increase (produced protein) Production Increase by Activity FVIII-Fc / / substantially enhances the production of Factor VIII-Fc

12 Platform: Fc Fusion of higg4 12 TNFα Binding Ability (ELISA) 10 Fold increase TNFRI-Fc (IgG1) TNFRI-Fc (IgG4) enhances the production of both types of Fc fusion protein

13 works on difficult proteins a1at-fc TNFRI-Fc IL13Ra2-Fc a1atmt-fc Original Secretion of Targeted Protein Maximum Effect Effect of may rescue problem proteins from misfolding and maximize secretion levels

14 Characterization Protein Aggregation Experiment conducted by Major Pharma Partner in Pilot Project PD group wanted to know whether can also results in lower aggregation. reduces the aggregation of produced proteins

15 Transition from 293T to CHO suspension system Protein Production in CHO Suspension conducted by CRO IL13Rα2-Fc Target Protein (Conditioned Media) Fold Increase (%) IL13Rα2-Fc enhances the production in CHO Suspension Expression System

16 Application for Screening R&D GPCR proteins (POC) receptor receptor receptor) receptor receptor enhances the expression of difficult-to-express proteins such as GPCR proteins

17 Application for Microbial Expression Soluble protein (SP) bacteria Bacterial Genome We customized platform for bacterial expression system by using a proprietary algorithms and intensive search for specific amino acid sequences involved in protein folding. plasmid Inclusion Body (IB) Test Protein A Total SP IB Platform Total SP IB Total SP IB Total SP IB Test Protein B Total SP IB Total SP IB Test Protein C increases both expression and solubility of the target protein in bacteria

18 Summary Advantages of Target proteins Increased Productivity by (%) 293T monolayer System 2-Fc protein 2000% 480% Factor FVII-Fc 1000% Factor IX-Fc 320% Factor VIII-Fc 2100% Avastin Humira 420% 600% 730% 293T Suspension System 2-Fc protein * 1100% CHO Suspension System 2-Fc protein * 400%