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Website antibodies

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  • VS2010_12_03 AX10_07
  • Transcript

    • 1. Ab-XTEN & Ab-XTEN-Drug Conjugates XTEN • Long, tunable PK enables up to once-a-month dosing Difficult to conjugate • Long PK maximizes efficacy and minimizes toxicity Hydrophobic Low Drug Load • Clinically validated in 2 products Fc-receptor mis-targeting ADCC/CDC • Non-immunogenic Bivalent • Biodegradable (safer than PEG) Monospecific No EPR effect • 3 publications 4 protein chains Ab-XTEN • Genetic fusion to Ab fragments & mimetics Easy, specific conjugation Highly hydrophilic • scFv, dAbs, Darpins, Nanobodies, Centyrins, Adnexins, etc. High Drug Load • Single protein chain, manufactured in E. coli No FcR mis-targeting No ADCC/CDC Mono- or Multi-valent Ab-XTEN-Drug Mono- or Multi-specific EPR effect • Specific conjugation to cytotoxic drugs via free thiols 1 protein chain • Many available drugs to choose from • Drug release via linker cleavage or XTEN degradation1
    • 2. AbFr-XTEN: Product Formats Monovalent AbFr IgG Drug Conjugate Target binding Cytokine Blockers Receptor Blockers Tumor-Drug Delivery Antiviral Bispecific AbFr C1 Binding Multifunctional Fusion Recycling ADCC Tumor-Specific Toxin Tumor-Specific IL2 Combination Products Immune activators • Large number of product opportunities • Common discovery platform2
    • 3. XTEN Prevents Aggregation of scFv Abs (215 nm) Size Exclusion Chromatography Monomer aHer2 Dimer Volume (ml) XTEN stabilizes the scFv and thus enables clinical use of scFv3
    • 4. Bispecific scFv-XTEN-scFv EGFR Her2 SDS/PAGE 160 aEGFR aHer2 110 1.0 Normalized Absorbance 60 Normalized Abs. (OD405 nm) GFP 0.5 20 Control 10 0.0 0.0001 0.01 1 100 10000 bscFc Concentration (nM) Versabody Concentration (nM) • High-affinity, specific binding to EGFR and Her2 • Single protein chain manufactured in E. coli4
    • 5. AbFr-XTEN Multimers Binding site: anti-EGFR Vhh Linker: 144AA Monomer Tetramer Hexamer Dimer Monomer 220 kD 160 kD Dimer 110 kD 80 kD Tetramer Hexamer Single Chain, multivalent, half-life, expressed in E. coli cytoplasm5
    • 6. AbFr Multimers are Well-Behaved EGFR Binding 1.6 B SA HRP Analytical SEC Profiles V Strept-HRP V EGFR-Fc 44 kDa 17 kDa 660 kDa 160 kDa 1.3 kDa 1.2 ELISA Signal GFP Anti-GFP 0.8 1 STD Monomer 0.4Normalized A215 Dimer 0.75 Tetramer Hexamer 0 0.5 Control Monomer Dimer Tetramer Hexamer 25 0.25 Plasma Stability 0 10 15 20 Days 0 1 3 7 Retention Volume (ml) VHH hexamer 50% pooled cyno plasma in PBS, 37C, Anti-GFP-HRP detection AbFr Multimers show avidity, no aggregation and good plasma stability6
    • 7. Chemical Conjugation to scFv XTEN medium short medium long SEC short long 44kDa 2 5 2 5 2 5 158kDa Absorbance 680nm 670kDa short med long aHer2 aHer2 aHer2 Elution Volume XTEN allows control of hydrodynamic radius and half-life7
    • 8. Cell Binding of aHer2-XTEN-AF Isotype control Trastuzumab block XTEN288 XTEN576 XTEN864 Protocol: SKOV incubated with AF680-labeled scFv-XTEN fusion proteins Cell binding detected by FACS XTEN length has little influence on cell binding8
    • 9. Tumor Uptake of aHer2-XTEN 60 Protocol: SKOV tumor model Fluorescence (Fold Increase) 50 3 mice per group 40 Day 0: Inject labeled protein 24hr 30 48hr Day 1&2: In vivo imaging 20 10 0 Long Medium Short Herceptin Long-chain XTEN facilitates tumor uptake of aHer2 fragment9
    • 10. In Vivo Targeting IgG aHer2-XTEN Trastuzumab A1 A2 A3 A1 A2 A3 Tumor Inject labeled Heart protein 3 day Kidney left distribution Kidney right Image organs Liver 1 Liver 2 Lungs Protocol: SKOV3 tumor model, n=3 Spleen Proteins labeled with AF680 • aHer2-XTEN576 shows specific tumor accumulation • Trastuzumab shows significant background staining of normal tissues10