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3 Potelligent CHOK1SV Webinar Presentation Alison Porter, 2010 Lonza Biologics plc, Slough, UK

4 Disclaimer Certain matters discussed in this presentation may constitute forward-looking statements. These statements are based on current expectations and estimates of Lonza Group Ltd, although Lonza Group Ltd can give no assurance that these expectations and estimates will be achieved. The actual results may differ materially in the future from the forward-looking statements included in this presentation due to various factors. Furthermore, Lonza Group Ltd has no obligation to update the statements contained in this presentation. Note: All slides are incomplete without verbal comments. slide 4

5 Scope of Talk POTELLIGENT technology Introduction Benefits GS Gene Expression System and CHOK1SV Introduction Benefits Combining technologies: Potelligent CHOK1SV Performance of the Potelligent GS-CHO technology Growth and productivity it characteristics ti Product characteristics ADCC slide 5

6 POTELLIGENT Technology

7 POTELLIGENT Technology Technology that applies an intelligent approach to creating more potent antibodies 100% fucose-free antibodies uses a proprietary fucosyltransferase-knockout (FUT8-KO) CHO cell line as a production cell Fucose-free mabs have markedly higher antibody-dependent dependent cellular cytotoxicity (ADCC) than fucosylated mabs Eight POTELLIGENT mabs in clinic Licensed by BioWa Intellectual properties of POTELLIGENT technology: Title PCT US Method of Modulating The Activity of Functional Immunological Molecules WO00/61739 A1 (Priority date 04/09/99) US7,214,775 (US2005/ ) EP A1 covers composition o of matter of fucose-free ee Mabs Antibody composition-producing cell WO02/31140 A1 US6,946,292 covers FUT8-KO mammalian cells (US2003/ ) EP A1 slide 7

8 POTELLIGENT mabs in Clinical Studies Compounds Target Ag Indication(s) Preclinical Phase-1 Phase-2 BIW-8405/MEDI-563 (KHK/MedImmune) KW-0761 (KHK/Amgen) IL-5R Asthma Immunology Allergy/ Immunology CCR4 Blood tumor / Oncology MDX-1401 (Medarex) CD30 Lymphoma MDX-1342 (Medarex) CD19 RA/CLL MDX-1411 (Medarex) CD70 RCC Oncology Immunology / Oncology Oncology Undisclosed - - Undisclosed - - BIW-8962 (KHK) GM2 Blood tumors Oncology slide 8

9 Summary of Clinical Data for KW-0761 and MEDI-563/BIW-8405 Antibody Target Molecule Cell Dose KW-0761 CCR4 ( /cell) Th2 cell ATLL/PTCL mg/kg mg/kg MEDI-563/ BIW8405 IL5Rα (<10 3 /cell) Eosinophil mg/kg Not immunogenic No adverse events relating to POTELLIGENT technology Highly active High antigen expression will not be required Active in blood, bone marrow, lymph nodes, and skin slide 9

10 POTELLIGENT and ADCC : N-acetylglucosamine : bisec GlcNAc : Mannose : Galactose : Sialic acid : Fucose Relationship between ADCC and structure of N-linked oligosaccharide id of antibody 1. Boyd et al. Mol.Immunol.1995: Sialic acid No effect 2. Kumpel et al. Hum. Antib. Hybrid. 1994: (+) Gal 2 to 3-fold increase of ADCC 3. Umana et al. Nat. Biotech. 1999: (+) BisecGlcNAc 10-fold increase of ADCC 4. Shinkawa et al. JBC J.B.C. 2003: POTELLIGENT (-) Fuc more potent ADCC than (+) Gal or (+) BisecGlcNAc Fucose Asn 297 slide 10

11 POTELLIGENT and ADCC Target Cell Antibody IgG1 ADCC FcγRIIIaγ NK Cell Enhanced ADCC has been confirmed by multiple antibodies Rituximab anti-t cell Mab Cytotoxici ity (%) mab concentration (µg/ml) POTELLIGENT mab Fucosylated mab Effector: human PBMC 37 C, 4hr incubation, LDH or Cr 51 release assay slide 11

12 Why Use the POTELLIGENT Technology Dramatically enhanced potency and efficacy of mabs Clinical results with POTELLIGENT -enhanced mabs expected to show higher efficacy in human patients when compared with nonenhanced antibodies Increased Fc receptor binding Overcomes the problem of low clinical responses due to genetic differences in the Fc receptor Lowered effective therapeutic dose of your antibodies Potential to deliver much more with much less mabs that are expected to be proven safe and well tolerated, with no immunogenic concerns Requires no change in current manufacturing process BioWa s proprietary technology works seamlessly with systems and processes already in place slide 12

13 GS and CHOK1SV

14 Industry-leading GS Gene Expression System Owned and licensed by Lonza A mammalian gene expression system For therapeutic proteins and monoclonal antibodies CHOK1SV GS-CHO and GS-NS0 cell lines Consistent and reliable means of Widely accepted generating high yielding cell lines Over 100 companies, 75 academic institutions Over 50 products in the clinic Speed 100s of high-yielding cell lines Amplification not usually created necessary (last 15 years) Stable cell lines Regulatory acceptance 7 Licensed products slide 14

15 CHOK1SV Adaptation of CHO cells to single cell suspension culture and to proteinfree medium can be problematic and time consuming One solution to this is to pre-adapt the host cell line to the desired culture conditions CHOK1SV is a suspension variant of CHO-K1 which has been preadapted to chemically-defined, animal component-free (CDACF) medium CDACF media can be used throughout all stages of the cell line construction process Compatible with single-cell cloning by FACS or other automated methods Cell lines derived from CHOK1SV exhibit excellent growth characteristics Reach high h maximum viable cell concentrations ti Able to maintain cultures at high viability slide 15

16 Comparison of Growth of Recombinant Cell Lines Derived From Different CHO Host Cell Lines 120 Viable Via able cell cell concentra ation ( /ml) / ml) Time (h) 22H11 LB01 22H11 created using CHO-K1 LB01 created using CHOK1SV Evaluated same fed-batch CDACF process in 10 L fed-batch bioreactor LB01 growth profile representative of CHOK1SV-derived cell lines slide 16

17 Comparison of Product Concentration from CHOK1SV and CHO-K1 Derived Cell Lines in 10 L Fed-batch Bioreactor Host cell line Xv max IVC Qp mab 10 6 cells /ml 10 6 cells /ml.h pg/cell.h mg/l CHO-K CHOK1SV Switch from CHO-K1 to CHOK1SV gave 3 to 4-fold increase in product concentration by increasing both Qp and IVC CHOK1SV Process improvements have given a further 4-5 fold increase in product concentration Product concentrations up to 9.5 g/l seen for alternative recombinant GS-CHO cell lines in latest process slide 17

18 Combining Technologies: Potelligent CHOK1SV

19 Potelligent CHOK1SV Potelligent CHOK1SV combining: BioWa s POTELLIGENT technology to generate more potent mabs through elimination of core fucose - With Lonza s GS Gene Expression System for robust manufacture of therapeutic antibodies Potelligent CHOK1SV is available for licensing through a threeparty agreement among BioWa, Lonza and licensee Potelligent CHOK1SV will be offered as part of Lonza's development services in combination with Lonza's GS Gene Expression System slide 19

20 Developing Potelligent CHOK1SV Three CHOK1SV derived host cell lines, whose alleles of FUT8 genes were completely disrupted, established Host cell lines were transfected with GS antibody vector As expected, antibodies produced from transfectants derived from the host cell lines show enhanced ADCC activity Cyto toxicity (%) Ab concentration (μg/ml) Effector: human PBMC Target: B-cell lymphoma Raji E/T ratio = 20:1 37 C, 4hr incubation LDH release assay CHOK1SV Potelligent A Potelligent t B Potelligent C slide 20

21 Developing Potelligent CHOK1SV - Continued One host cell line selected for further development Potelligent C Based on growth and productivity data following transfection of the 3 host cell lines Number of Cell Lines Assessed in 24-well plates Product Concentration (mg/ L) Assessed in batch shake-flask culture Number of Cell Lines Product Concentration (mg/ L) Potelligent A Potelligent B Potelligent C Potelligent A Potelligent B Potelligent C Approximately 60 cell lines from each host Approximately 30 cell lines from each host slide 21

22 Developing Potelligent CHOK1SV - Continued Lead host cell line named Potelligent CHOK1SV Full cell line construction undertaken using Potelligent CHOK1SV Including assessment of recombinant cell lines in lab-scale l bioreactors ADCC assay on antibody samples from bioreactors cgmp Master Cell Bank (MCB) and Working Cell Bank (WCB) prepared Including regulatory data pack to support Potelligent CHOK1SV slide 22

23 Performance of Potelligent GS-CHO Technology

24 Growth and Productivity Characteristics: Fed-batch Shake-flasks (Antibody 1) Individual Value Plot of Max. the Maximum Viable Cell Concentration Achieved in Fed-Batch Shake-Flasks Maximum 10 6 viab Xv ble (x10e6 ( cells/ml cells/ml) Potelligent CHOK1SV CHOK1SV IVC 10 9 (10E viabl 6 le 9 cells.h/l) Individual Value Plot of the IVC IVC Achieved in Fed-Batch Shake-Flasks Potelligent CHOK1SV CHOK1SV Individual Value Plot of Product Concentration Achieved in Fed-Batch Shake-Flasks Product t Concentration mg/l (mg/l) Individual Value Plot of Specific Production Production Rate Achieved Rate in Fed-Batch Shake-Flasks Potelligent CHOK1SV CHOK1SV Potelligent CHOK1SV CHOK1SV Qp (pg/cell/h) *Research with antibody 1 sponsored by KaloBios slide 24

25 Growth and Productivity Characteristics: Fed-batch Shake-flasks (Antibody 2) Individual Value Plot of Max. the Maximum Viable Cell Concentration Achieved in Fed-Batch Shake-Flasks Maximum 10 Xv 6 cells/ml (x10e6 cells/ml) ) Potelligent CHOK1SV CHOK1SV Individual Value Plot of Product Concentration Concentration Achieved in Fed-Batch Shake-Flasks 2500 Produc ct Concentration mg/l (mg/l) Potelligent CHOK1SV CHOK1SV IVC 10 9 (10E9 viabl e cells.h/ml) cells.h/l Individual Value Plot of the IVC IVC Achieved in Fed-Batch Shake-Flasks Potelligent CHOK1SV CHOK1SV Individual Value Plot of Specific Production Production Rate Achieved Rate in Fed-Batch SHake-Flasks 2.0 Qp (pg/cell/h) Potelligent CHOK1SV CHOK1SV slide 25

26 Growth and Productivity Characteristics: 10 L Fed-batch Bioreactor Cultures (Antibody 2) Viable ce ell concentration (10 6 /m ml) Growth Characteristics Antibody concentra ation (mg/l) Elapsed time (h) Elapsed time (h) Productivity Characteristics Key: Red line = Potelligent CHOK1SV derived cell lines Blue line = CHOK1SV derived cell lines slide 26

27 Growth and Productivity Characteristics: 10 L Fed-batch Bioreactor Cultures (Antibody 2) - Continued Host Cell Line Potelligent CHO OK1SV IVC at Harvest 10 9 cell.h/l Viability at Harvest (%) Overall Production Rate (Qp) pg/cell/day Antibody Concentration at Harvest mg/l CHO OK1SV Successful identification of Potelligent CHOK1SV derived cell lines, with suitable growth and productivity characteristic, for use in a manufacturing process slide 27

28 Product Characteristics: Reduced + Non-reduced SDS PAGE and IEF 10 L Fed-batch Bioreactor Cultures (Antibody 2) Reduced Non-reduced Reduced and non-reduced SDS PAGE Lanes 4 to 7: Potelligent CHOK1SV Lanes 8 to 9: CHOK1SV Comparable band profiles for all samples IEF Gel on left, lanes 4 to 7: Potelligent CHOK1SV Gel on right, lanes 2 to 3: CHOK1SV Comparable band profiles for all samples slide 28

29 Product Characteristics: GP-HPLC 10 L Fed-batch Bioreactor Cultures (Antibody 2) Relative Peak Intensity (%) Host Cell Line Total Aggregate Monomer Fragment Potelligent CHOK1SV <0.1 CHOK1SV <0.1 GP-HPLC Aggregate levels in acceptable range (0 5%) Comparable aggregate and monomer levels slide 29

30 ADCC Activity of Antibody 2 Produced from 10 L Fed-Batch Bioreactor Cultures Effector: human PBMC Target: B-cell lymphoma Raji 37 C, 4hr incubation, LDH release assay Potelligent CHOK1SV CHOK1SV Potelligent CHOK1SV produced antibodies with enhanced ADCC as expected slide 30

31 Summary Introduction of a new host cell line, combining Lonza and BioWa owned technologies Potelligent CHOK1SV cgmp MCB and WCB available Recombinant cell lines created using Potelligent t CHOK1SV have shown Enhanced ADCC Growth suitable for a production process Further optimisation i i possible High product concentration levels in a platform process Further optimisation possible Work seamlessly with current manufacturing process Potelligent CHOK1SV is available for licensing through a threeparty agreement among BioWa, Lonza and licensee Potelligent CHOK1SV will be offered as part of Lonza's development services in combination with Lonza's GS Gene Expression System slide 31

32 Acknowledgements Lonza Samantha Vieira Tarik Senussi Alison Mastrangelo Gavin Edwards Andy Racher BioWa/Kyowa Hakko Kirin Naoko Yamane-Ohnuki Shigeru Iida Mitsuo Satoh Kenya Shitara Masamichi Koike slide 32

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