Digital Display Libraries using High Density Peptide Arrays Max Bergmann Konferenz XXXVI

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1 Digital Display Libraries using High Density Peptide Arrays Max Bergmann Konferenz XXXVI Klaus-Peter Stengele, Jigar Patel Roche NimbleGen Inc, Madison, WI

2 Roche NimbleGen Peptide Array Spatially ordered chemistry by light-directed synthesis Digital Micromirror Device 2 2

3 NimbleGen Peptide Array Platform Spatially ordered smart light synthesis Comprehensive/Unbiased Represent entire Human Proteome or other Proteomes on 1 slide/assay Represent a wide diversity of random/comprehensive peptides for binder discovery Natural and unnatural aminoacid (D-amino, Citrulline, HomoCitrulline, Methyl Amino, Peptoids etc) Linear or cyclic peptides Currently 2.9M peptides per array, expandable to > 7M Consistency High reproducibility due to digitallycontrolled chemical synthesis 3

4 Read the Immune system Synthesize every peptide in every protein Protein Antibodies bound to peptides serum Peptide Reactive Epitopes / Biomarkers 4

5 Raw Peptide Array Image (Human Proteome, 2.9M peptides) Alignment and QC controls (Streptavidin binding peptide)

6 Molecular & Cellular Proteomics 13: , , High reproducibility Epitopes consists of, α helical elements β-pleated sheets loops

7 Peptide Binder Discovery using Directed Evolution - platform (mrna display, Phage display, combinatorial synthesis etc) - diversification (natural / non-natural amino acids, cyclization) - selection (binding, catalytic agonist / antagonist, allosteric, PPI s) - amplification (depends on method, lead optimization)

8 88mer library Selection of binder to Streptavidin HPQ

9 Peptide libraries can be enormous What is the optimal length of a peptide with medical value? No. of amino acids Library size Library weight (only one molecule of a kind) x mg x ,350 kg x x10 24 kg Earth mass = 6 x kg

10 Binder discovery using comprehensive 5-mer (+) array To represent all 5-mer L-aminoacid peptides = 20^5 = 3.2M peptides but exclude cysteine = 19^5 = ~2.47M peptides = spots / array Synthesize all unique 2.47M 5-mer peptides, flanked by a wobble synthesis mix and linkers at each end Z Flexible soluble linker G:S (3:1) J Mixture of all 20 amino acids [ZZZZJ] 5mer [JZZZZ] (Pseudo 7mer) = 40 different peptides / spot 100 mln peptides represented Synthesis + Deprotection: 8 hrs Target Binding: Streptavidin Cy5 for 1 hr Washing: with 1X TBST buffer for 5 mins Scan: 2 um ~ 40 mins Extract peptides with highest fluorescent signal, for clustering analysis

11 Top 200 reactive peptides to Streptavidin H P Q A G F V XX HPQ [AGFV] - XX

12 Extension of Core Peptides by 4 aa (2aa N-term and 2aa-C-term) XX HPQ [AGFV] - XX XXHPQAXX XXHPQGXX XXHPQFXX XXHPQVXX 160,000 peptides 160,000 peptides 160,000 peptides 160,000 peptides Top 100 peptides Top 100 peptides WQHPQAGK WDHPQGGR WTHPQFQK WDHPQVGG Single and Double AminoAcid Maturation

13 Systematic Maturation and Optimization ATHPQFQK CTHPQFQK DTHPQFQK ETHPQFQK FTHPQFQK GTHPQFQK HTHPQFQK ITHPQFQK KTHPQFQK LTHPQFQK MTHPQFQK NTHPQFQK PTHPQFQK QTHPQFQK RTHPQFQK STHPQFQK TTHPQFQK VTHPQFQK WTHPQFQK YTHPQFQK ATHPQFQK AAHPQFQK ATAPQFQK ATHAQFQK ATHPAFQK ATHPQAQK ATHPQFAK ATHPQFQA ACHPQFQK ATCPQFQK ATHCQFQK ATHPCFQK ATHPQCQK ATHPQFCK ATHPQFQC ADHPQFQK ATDPQFQK ATHDQFQK ATHPDFQK ATHPQDQK ATHPQFDK ATHPQFQD... Alanine Scan Cysteine Scan Glutamic Acid Scan All 20 amino acid scan Or introduce non-natural aa

14 Streptavidin binding to peptides with single amino acid substitution W T H P Q F Q T A Del

15 In Summary: Rapid, stepwise discovery and maturation of peptide binders 5-7 mer CORE Core Peptide Binder Core Hotspot Peptide (2) + (5-7) mer + (2) N-term C-term extension with 2mer+ array 2mer extension on N+C terminal (2) + (9-11) mer + (2) N-term C-term extension with 2mer+ array 2mer extension on N+C terminal 5-15 mer Peptide maturation of extended peptide Maturation and final optimization 15

16 Summary Multiple peptide binders / binder families can be identified systematically and evolved to identify the hotspot binding region Hotspots for linear peptides range from 3-9 mer Identified HPQ containing peptide that binds to streptavidin Discovered > 4 novel peptide binders for streptavidin, each evolved independently, not HPQ Screens can be performed using cyclic peptides, and/or D-amino acid or other variants Next generation synthesizer can have 100 building blocks on-board Binder selection can be performed with different linker types, variable ph, temperature depending on the target of interest Multiple targets already tested, eg PSA, TNF-alpha, BACE, cross-mab Average turn-around time on the order of less than 1 month No project failure to date

17 Acknowledgements Roche NimbleGen Roche Professional Diagnostics Rebecca Selzer Ryan Bannen Todd Richmond Kurt Heilman Leslie Lincoln Alan Pitas Eric Dauenhauer Jon Voichick Thomas Albert Victor Lyamichev Eric Sullivan Jeffrey Jeddeloh Dieter Heindl Frank Bergmann Michael Schraeml Frank Kroner 17

18 Doing now what patients need next

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