Sutro Biopharma Inc. Xpress CFTM: A rapid platform for drug development from antibody discovery to manufacturing August 2014 Sutro’s Xpress CF™ Platform: Cell-Free Synthetic Biology input (DNA) Assembled IgG + Energy + Accessories* (AA, proteins, RNAP, metabolites) Ribosome (Catalyst) output Engineered cell-free extract (multi-domain (E. coli) eukaryotic proteins) CONFIDENTIAL *To be incorporated into strain for commercial production 2 Advantages of Sutro’s Xpress CF™ Platform • Protein production is separated from biomass, allowing – – – – Off-the-shelf protein production Rapid expression for short make/test cycle Expression of toxic proteins high titer expression at g/L scales • Xpress CFTM is an “open” system that can be manipulated, allowing – Easy incorporation of non-natural amino acids – Biochemical optimization of proteins via additives • An ideal platform for antibody discovery! CONFIDENTIAL 3 Engineered extracts for improved IgG folding IgG CONFIDENTIAL 4 Rapid Execution of Antibody Discovery Programs Discover novel antibody fragments using ribosome display and screening Express antibodies and fragments with Sutro protein synthesis system Reformat fragments in host of different bispecific and antibody frameworks Select the best lead candidate based on in vitro and in vivo activity 5 Ribosome Display Antigen-biotin Antibody Streptavidin capture Ribosome PCR amplify mRNA + Ag No Ag Screen output CDR H3 Ribosome Display • Ribosome Display is a selection technology well-suited for CF • Built-in bias for variants that express and fold well in CF • Selection output translates directly into CF-based screening Round 1 Round 2 Round 3 Round 4 CONFIDENTIAL 6 Source of Antibodies and Integration into the Sutro System • • • • • Mouse Rabbit Chicken Humanized Mice Human B-cells B-Cell Based Technologies Phage Display Bacteriophage Display CellBased Display Ribosome Display Bispecifics Many Scaffolds • Bacteria Display • Yeast Display • Mammalian Display Sutro Cell Free Antibody Discovery Antibody Engineering Sutro System nnAA Linkerwarhead ADCs 7 Sutro’s Libraries For Antibody Discovery Method Format Framework Diversity Size scFv Optimized Human Germline Synthetic >1012 Fab Optimized Human Germline Consensus Synthetic >1012 scFv Optimized Human Germline Synthetic >109 Fab Naïve Human Immune Repertoire Natural >109 Ribosome Display Phage Display High Throughput Screening Capabilities Biacore Affinity Ligand blocking Selection (1012) Primary Screen (1000’s) Secondary Screen (100’s) ELISA Cell binding or internalization Epitope Binning CONFIDENTIAL 9 Screening workflow Throughput/week 1000’s Colony picking DNA prep Cell-free reaction & ELISA-based ranking 100’s Flower Plate Expression Oasis 600 100’s 10 ELISA and FACS binding Ligand Blocking Biacore kinetic analysis Detailed characterization of variants HT purified protein Protein quantitation ELISA And Cell Binding Kinetic characterization CONFIDENTIAL Epitope binning Cell Killing (secondary) 11 Ribosome Display: unique platform for antibody discovery and optimization +Ag -Ag Rd1 Rd2 KD (nM) Rd3 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 Parent Improved clones New lead Parent 0 500 1000 1500 2000 Recovered protein (ug/mL) KD vs protein recovered Cell Binding CONFIDENTIAL ELISA 12 Antibody leads can be combined in multiple bispecific scaffolds scFv scFv1scFv2 scFv1-scFv2- Fc scFv1-Fc-scFv2 scFv-scFc scFv-Fc scFv1-scFc-scFv2 Rapid expression of multiple scaffolds to define product format with optimal activity and half-life 13 Rapid Screening of Bispecific Combinations A A HIK B B A A Hole x stumpK knob CONFIDENTIAL 14 Bispecific-bridging of 2 targets with AlphaLISA HIK A B A B CONFIDENTIAL KIH 15 Translation of nnAA-Containing Proteins Enables Site-Specific Conjugation input (DNA) nnAA CF Engineered MJ TyrRS + NNN tRNA mRNA Stop NNN UAA Ribosome (Catalyst) cell-free extract (E. coli) Sutro Azido nnAA IgG with DBCO-Based Linker-Warhead Azido nnAA Cu Free Click Conjugation Chemistry DBCO Linker-Peptide Warhead (MMAF) Data Driven Design: Production of Many Variants in Hours SP Number Position TAG site o Mutate Sites in IgG: Choose nnAA sites using rational design, or just make all of them! o Produce nnAA IgG: Incorporate nnAA at 100’s of chosen sites o Conjugate: Conjugate nnAA with appropriate chemistry o Purify: Separate conjugated IgG away from unincorporated linker-warhead o Test: Assay conjugated IgG’s for binding and cell killing Light Chain: 111 Sites Heavy Chain: 133 Sites 18 Rapid Selection of Optimal Sites for nnAA Incorporation 1500 1000 nM 500 DAR 0.4 0.7 1.1 DAR 1.1 1.5 1.6 150 150 0 0.01 0.1 Relative Cell Viability (Cellular ATP content, % of control) Conjugation Efficiency (Drug/MAb Ratio) Transform of Transform of Data 1 Data 1 100 50 Relative Cell Viability MFI 2000 Herceptin Sutroceptin E293 K334 Viability Relative (Cellular Cell ATP content, % of control) 2500 Mean Fluorescence Intensity SKBR3 Binding Assay Conjugated Variants Compared to Herceptin nnAA Incorporation and Expression 0 0.0001 Cell Killing Assay 1 10 100 1000 T359 T359 K360 N361 Q362 K360 K370 Y373 S375 W381 K370 Y373 S375 W381 S383 S383 CF-Trastuzumab N361 nM 100 50 Q362 Pos N384 Cont ADC N384 S136, GY S136, GY DAR=1.6 WT HIC WT HIC 0 0.0001 0.001 S136 ERB2 S136 ELISAERB2 E 0.001 0.01 ug/ml 0.010.1 ug/ml mg/mL 0.1 1 1 Best Single Site ADCs Show Differential Efficacy (single 15 mg/kg IV dose) 1600 Free Drug, 0.54mg/kg Dose equivalence at DAR of 4.0 Tumor Volume, mm3 1400 Vehicle 1200 HC Site 1, 15mg/kg 1000 Unconjugated control HC Site 1, DAR 1.84 15mg/kg HC Site 4, DAR 1.97 Trastuzumab-CF AB4285 (MMAF) HC Site 5, DAR 1.97 800 600 400 HC Site 6, DAR 1.96 200 0 0 10 20 30 40 Days 50 60 70 HerceptinTM, 1x 30mg/kg (t =0), 3x15mg/kg (weekly) All MMAF ADC treated groups vs. vehicle p < 0.0001**** up to d45 • • • All treatments are single dose i.v. @ t=0 Multi-doses of HerceptinTM dosed i.p No significant weight loss observed in all treatment groups Xpress CF™ High Titers at Multiple Scales 21 Scalable and Efficient a [rhGM-CSF], mg/L 800 250 uL 300 mL 600 4L 100L 400 200 0 0 200 400 600 Time, min Zawada et al, (2011) Biotech. & Bioeng. Rapid Production of Biotherapeutics 2 DAYS 4 6 8 Sutro Technology Synthetic DNA • • • • 50 µ g protein HTS plate format 5g protein 5-L reactor 100 g protein 100-L reactor Direct linear scale-up from HTS to production scale Uses standard bioreactors & downstream equipment Minimal, rapid process development Gene sequence to drug substance in days Speed to the Clinic …
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