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ThioBridge™ - Next generation conjugation technology for ADCs Antony Godwin Director of Science & Technology Antibody Drug Conjugates: Innovation in Design, Development and Manufacture June 26, 2013 Insert appropriate image Presentation Overview • Company Overview • Conjugating To Antibodies – The Challenges • ThioBridge™ Thiol Bridging Conjugation • Case Studies With mAb And Fab 2 2 Company overview • Comprehensive portfolio of proprietary technologies Pharmacokinetic optimisation of biologics via sitespecific polymer conjugation Superior antibody-drug conjugate technologies for reduced heterogeneity and increased stability • Business model offers product-specific technology licences following feasibility studies and collaborative programs Multiple partner programs progressing towards clinic • 45 employees, including 26 PhDs, based in dedicated lab and office facilities in central London and Coventry 3 3 Enabling Better Biopharmaceuticals Antibody Drug Conjugates (ADCs) 4 Less heterogeneity Improved stability Efficient chemistry Range of payloads mAbs, Fabs, scaffolds Optimisation of halflife & activity Bispecific & novel products Site-specific conjugation Low reagent use Novel polymer for low viscosity conjugate Chemical conjugation to rapidly produce • homodimers • heterodimers • bispecific products Leading the way - Approved ADCs Maleimide linkage not direct to mAb Kadcyla (T-DM1, Roche/ImmunoGen) FDA approval 2013 Amine conjugation Adcetris (Seattle Genetics/Millennium) FDA approval 2011 Average DAR 3.5 Disulfide breaking thiol conjugation Maleimide linkage to mAb Based on amine and disulfide breaking conjugation 5 Average DAR 4 Payload conjugation to a mAb has challenges: There is a need to address heterogeneity & stability Lysine chemistry • Low efficiency • Non-specific • High heterogeneity 6 Cysteine chemistry • Semi-specific • Bond breaking • High heterogeneity • Maleimide instability Cysteine chemistry • Re-engineering approach • Site-specific • Reduced heterogeneity • Maleimide instability ThioBridge™ attaches payload across a native disulfide bond that is re-bridged Accessible disulfide bonds: 7 mAb Fab Bridging conjugation is widely applicable 8 Category Example protein / peptide Enzymes Asparaginase Amylase Cytokines Interferon α-2a and α-2b Interferon β Hormones Leptin Erythropoietin Peptides Tumour imaging ligand Blood proteins Coagulation factors Antibody fragments Anti-CD4 Fab Anti-TNF α domain Novel formats Scaffolds Activity ThioBridge™ S-S bridging reagent • • • • Combined with payload in single reagent Compatible with amine and hydroxyl functionalised payloads Cleavable and non-cleavable linker options Hydrophilic for improved solubility/reduced aggregation Thiol bridging linker 9 Hydrophilic spacer Degradable or Nondegradable linkers Amenable to payloads with different MOA’s ThioBridge™ ADCs are more homogeneous More homogeneous DAR distribution restricted to up to DAR 4 – can adjust distribution of DARs 2, 3 & 4 Avoids highly loaded species Minimal amounts of non-conjugated antibody DAR2-4= 94% DAR2-4= 52% % 32% 0% 012345678 012345678 ThioBridge™ Maleimide conjugation to reduced disulfide bonds Hamblett et al. Clin Cancer Res (2004) 10 DAR=0 DAR=1 DAR=2 DAR=3 DAR=4 DAR=5 DAR=6 DAR=7 DAR=8 • Competitive inhibitor • • • • Lower tolerability Accelerated clearance High instability High hydrophobicity Instability of maleimide conjugates: Deconjugation reactions antibody conjugate albumin in serum (A) albumin conjugate Maleimide instability can lead to conjugation of drug to albumin e.g., Senter et al 11 ThioBridge™ conjugates are more stable in serum than maleimide • Alexa Fluor 488 labelled trastuzumab • ThioBridge™ & Maleimide conjugates incubated in rat serum at 37 °C for 48 h • SEC analysis Relative Fluorescence ThioBridge™ conjugate Maleimide conjugate Significant crossconjugation to albumin T=0h T=0h mAb unstable Aggregates T = 48 h 4 6 Time (min) Alexa Fluor 488 T = 48 h 8 10 12 14 mAb Albumin 16 18 20 4 6 Time (min) 8 10 12 14 mAb Albumin Significant instability seen with maleimide conjugate but not with ThioBridge™ conjugate 12 Fragments 16 18 20 ThioBridge™ is more stable than maleimide in the presence of human serum albumin • Same payload (Drug) conjugated to trastuzumab using ThioBridge™ and maleimide • Both conjugates were incubated in artificial serum (PBS + 20 mg/mL HSA) at 37 °C 4 2 3.8 1.9 3.6 1.8 3.4 1.7 3.2 Maleimide ADC (DAR = 4) 3 2.8 ThioBridge™ ADC (DAR = 4) 2.6 Average DAR Average DAR • HIC was used to determine changes in the average DAR 1.6 1.4 1.2 2.2 1.1 2 1 50 100 ThioBridge™ ADC (DAR = 2) 1.3 2.4 0 Maleimide ADC (DAR = 2) 1.5 0 Time [hours] Superior stability in artificial serum for ThioBridgeTM compared with maleimide 13 50 Time [hours] 100 ThioBridge™ ADC shows excellent and antigen-selective cell-killing in vitro Antigen-positive II Antigen-positive (BT-474) II (BT-474) Antigen-positive I (SK-BR-3) SK-BR-3 100 50 0 -2 -1 % viability % viability Antigen-positive cell lines % viability 100 BT-474 100 50 0 0 1 2 3 -2 4 -1 [ADC] (nM)-log scale Antigen-negative I (A549) ThioBridge ADC (DAR=2.8) 100 % viability % viability 5 MCF-7 50 0 Free drug Free drug Trastzumab Trastzum ThioBridg ThioBridge ADC (DAR=2.8) 50 0 -1 0 1 2 3 [ADC] (nM)-log scale 4 5 -2 -1 ThioBridge™ ADCs are c.10,000-fold more potent in antigen +ve cells than in antigen –ve cells 14 Trastzuma ThioBridge ADC (DAR=2.8) ThioBridg 0 1-2 2-1 30 41 52 3 4 5 0 [ADC] (nM)-log scale [ADC] (nM)-logIIscale Antigen negative (MCF-7) 100 -2 Free drug 50 A549 Antigen-negative cell lines Free drug drug Free Trastzumab Trastuzumab Free drug Trastzumab 0 1 2 3 [ADC] (nM)-log scale 4 5 ThioBridge™ ADCs are stable in serum • ThioBridge™ ADC was incubated at 37°C in human serum for 72 h prior to measuring the potency in antigen-positive (BT474) cells [ADC](nM) – Log scale ThioBridge™ ADC retains in vitro potency after incubation in human serum 15 ThioBridge™ ADC highly effective in BT474 xenograft model 24 Body weight Tumour volume 22 1600 20 Body weight (g) Tumour volume (mm3) 1400 1200 1000 800 600 18 Vehicle 16 mAb drug 14 Thiobridge ADC (30 mg/kg) ThioBridge ADC (20 mg/kg) 12 ThioBridge ADC (10 mg/kg) 400 10 1 200 1 5 9 13 17 21 25 29 33 37 41 13 17 21 25 29 33 37 41 45 45 Time (days) Dosing Schedule 9 Time (days) 0 vehicle / mAb /drug (i.v.) ThioBridgeTM ADC 10 mg/kg (i.v.) ThioBridgeTM ADC 20 mg/kg (i.v.) ThioBridgeTM ADC 30 mg/kg (i.v.) 16 5 Complete responses at highest ADC doses Sustained for at least 60 days Significantly better efficacy in ADC groups Well-tolerated at all doses ThioBridge™ is ideally suited for conjugation of antibody fragments (Fab) 17 Advantages of using ThioBridge™ to create ADCs using Fabs ThioBridge™ Fab-DC SDS-PAGE analysis of reaction mixture ThioBridge Drug conjugate 18 Site-specific conjugation Conversion nearly 100% Low reagent consumption Single reaction product (DAR = 1.0) Easy purification and characterisation 18 ThioBridge™ Fab drug conjugate 100% 100% 100% 100% Complete bridging of ThioBridge™ Fab drug conjugate and excellent purity 19 ThioBridge™ Fab and mAb ADCs internalise into antigen-positive cells • A fluorescent mock payload (FITC) was conjugated to trastuzumab Fab and mAb using ThioBridge™ • Internalisation into antigen-positive cells (SKBR3) was investigated by confocal microscopy ThioBridge™ mAb conjugate ThioBridge™ Fab conjugate ThioBridge™ Fab and mAb conjugates internalise efficiently into antigen-positive cells : ADCs obtained using a bis-alkylating reagent internalise into antigen-positive cells. (a) conjugate. (b) Fab-FITC conjugate. 20 20 ThioBridge™ conjugation of Fabs does not impair antigen binding • ELISA in reversed orientation (Fab / FabDC immobilised on the plate) • Detection with: • biotin-HER2 / streptavidin-HRP • 6His-HER2 / aHis-mAb-HRP ThioBridge™ Fab-DC retains high affinity binding to antigen 21 21 ThioBridge™ Fab drug conjugate shows potent and selective cell-killing in vitro Antigen-positive cell line Antigen-negative Antigen-negative cell cell line line ThioBridge™ Fab-DC Drug Drug [Fab-drug conjugate] (nM) – log scale % viability Fab ThioBridge™ Fab-DC % viability Fab [Fab-drug conjugate] (nM) – log scale In antibody positive cells, the activity of ThioBridge™ Fab-DC matches free drug No effect of ThioBridge™ Fab-DC observed in antigen negative cells 22 ThioBridge™ Fab drug conjugate highly effective in BT474 xenograft model Vehicle 23 Vehicle mAb (20 mg/kg) Drug (same dose as in Fab-DC) Fab-DC (20 mg/kg) Dosing schedule • q2d vehicle, free drug & ThioBridge™ Fab-DC • q4d mAb mAb drug Fab-DC Potential CMC advantages of using ThioBridge™ to create ADCs • Controlled DAR distribution (no greater than DAR 4) • Easier characterisation • Low reagent stoichiometry (near 1:1 stoichiometry) • Improved aggregation profile • Excellent stability 24 CONFIDENTIAL 24 Conclusions: • Improved homogeneity No re-engineering of mAb Site directed to disulfide bonds • Improved stability Re-bridged disulfide bonds Preserved structural integrity • PEG spacers Reduced aggregation ThioBridge™ Improved reagent solubility IP & partners Granted Patents Commercial Partners Half-life extension Antibody-drug conjugates Claims cover reagents, conjugation process and conjugate products 26 Feasibility studies and collaborations ongoing with non-disclosed partners Thank you for your attention Antony Godwin Director of Science & Technology [email protected]