Chris Ullman, Isogenica, 'The use of CIS display for drug discovery'
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Chris Ullman, Isogenica, 'The use of CIS display for drug discovery'
- 1. The Use of CIS Display for Drug Discovery p y g y Cresset European Users Meeting 2011
- 3. CIS Display Molecular Evolution Technique CIS display is an acellular, in vitro display technology • Uses biochemical process of E. coli • No cloning, therefore larger libraries (>1013) are rapidly generated • CIS display is a powerful discovery engine for peptides and other protein scaffolds other protein scaffolds
- 4. Molecular Evolution vs Molecular Evolution vs HTS Molecular evolution – 1010‐1013 per week HTS – 106 per week 4
- 5. Phage Display transformation DNA library preparation • Li k b Link between genotype and phenotype • Typically upto 1010 peptides but has disadvantages
- 6. Ribosome mRNA IVC CIS display display display Acellular emuls Technologies sion Expression of protein Expression of protein from an in vitro transcription/ translation mixture translation mixture CIS RepA Mattheakis et al, 1994 PNAS Nemoto et al, 1997 FEBS Lett. ori Roberts and Szostak, 1997 PNAS A Tawfik & Griffiths 1998, Nat. Biotech Doi et al, 1999 FEBS Lett. Odegrip et al., 2004 PNAS RNA template | DNA template
- 7. CIS Display Mechanism Promoter Library repA CIS ori Linear dsDNA template Nascent mRNA polypeptide Translating Translating ribosome RNA polymerase RNA polymerase Odegrip et al., 2004 PNAS, 2806‐2810 Promoter Library repA CIS ori Displayed polypeptide RepA Promoter Library repA CIS ori
- 8. CIS Display – Libraries to Active Candidates 1. Cis‐activity of DNA‐ binding protein RepA bi di i 2. Libraries expressed in vitro → stable protein‐ → stable protein DNA complexes CIS 3. Protein‐DNA display di l complexes subjected l bj d to affinity selection panning 4. . Eluted complexes uted co p e es regenerated by PCR 5. Optimal ligands after 3‐5 successive rounds 35 i d 6. Optimised with in‐ y house lysate
- 9. Next Generation Approach to Biologics Discovery CIS display Next generation selection sequencing QC In silico screening DNA tags Peptide tags Peptide tags Peptide output Peptide clusters Enriched peptides QC Screening QC Maturation
- 10. Next Generation Peptide Screening Platform CIS display peptide selection process monitored by Illumina NGS. Approximately 4 million sequences from panning rounds 1‐4 of Approximately 4 million sequences from panning rounds 1 4 of CIS display determined Percentage of sequences ≥0.1% (found at least ~4,000 times) f 0 1% (f d l 4 000 i ) selelection B Selection B 30 25 ences 20 % seque 15 10 5 0 R1 R2 R3 R4
- 11. Evolution of Top 20 Peptide Sequences Evolution of Top 20 Peptide Sequences Evolution of top 20 sequences through panning rounds 2‐4 Evolution of top 20 sequences through panning rounds 2‐4 Enrichment of individual sequences can be monitored in the process 14% 3.0% 12% 2.5% 10% 2.0% 8% 1.5% 6% 1.0% 1 0% 4% 2% 0.5% 0% 0.0% R2 R3 R4 R2 R3 R4
- 12. Bioinformatics Analysis Following CIS Display After 4 rounds of enrichment using CIS display from ~1013 sequences against client target: ~4.5x106 DNA sequences analysed in hours 14,000 peptides 447 clusters 197 enriched fragments
- 13. Next Generation Sequencing – In Silico Screening Bioinformatics Analysis Bi i f ti A l i Selection output: >6x106 sequenced clones FLAG‐tag: YKxxD
- 14. Peptide Screening – Anti‐TNFα Peptide Selection Library 1, 36‐mer; Library 2 and 3, biased sequences; Library 4, co st a ed a d 6 e constrained and 16‐mer linear mix. 3 different lysates. ea . 3 d e e t ysates. Selection generated high hit rate but difficult to determine consensus 15,526 16‐mer peptides from library 4 passed to PEPperPRINT for high‐ density peptide synthesis
- 15. Peptide Screening – Anti‐TNFα Peptides Following 4 rounds of enrichment using CIS display from >1013 sequences : • In collaboration with PEPperPRINT • Result: >15,000 16‐mer peptides synthesised in parallel • Binding motifs and better discrimination between specific and non‐specific between specific and non‐specific Powerful dataset P f ld t t • Library designs validated through peptide synthesis and binding • M tif i Motifs important for IP protection, t t f IP t ti therapeutics, epitopes • Firm basis for maturation libraries
- 16. Isogenica s Maturation Approaches Isogenica’s Maturation Approaches Sequential amino acid scan q (Single mutation) Consensus‐based library Consensus based library (Multiple changes but maintain important sidechains) Variable consensus library (Additonal conservative substitutions at consensus) Error‐prone library (Mutations at random positions) (Mutations at random positions) Fully doped library (mutations introduced at custom rates)
- 17. CIS Display Maturations • Using the speed of CIS display to build multiple maturation libraries – driving to optimal binders • Picomolar binders, best binder to date <20 pM originally selected clone improved peptides improved peptides 16‐mer peptide libraries
- 18. Maturation of Anti NGF Peptides Maturation of Anti‐NGF Peptides Initial selections identified NGF binding peptides Maturations have identified 2 candidate peptides that inhibit NGF binding to its high affinity receptor binding to its high affinity receptor TrkA involved in the pain response pathway The peptides inhibit a phenotypic change in rat PC12 cells (from p pheochromocytoma of the rat y adrenal medulla) in the presence of NGF NGF causes neurite outgrowth in rat PC12 cells. Peptides A2 and D9 p inhibit this change
- 19. Maturation of Anti NGF Peptides Maturation of Anti‐NGF Peptides peptide candidates inhibit receptor peptide candidates inhibit receptor interaction with high activity peptides have now been PEGylated peptides have now been PEGylated for in vivo studies (Polytherics Ltd.)
- 20. Valuable Outputs Valuable Outputs Potential to identification of binding sites and identify epitopes CIS display can provide an evolutionary approach to determine natural substitution matrices based upon a peptide determine natural substitution matrices based upon a peptide backbone Important sidechains are identified through consensus Important sidechains are identified through consensus sequences However, current clustering algorithms can be ineffectual – , g g sequence not shape New algorithms to cluster peptides based upon field g p p p characteristics
- 21. BIC Platform Partners BIC Platform Partners Isogenica Biolauncher Cresset Group Very large diverse Novel representation Proven field based peptide libraries tid lib i of peptide binding f tid bi di chemistry force fields h it f fi ld Intracellular and populations Expert Peptidomimetic extracellular targets Cluster binding design Harnesses NGS to id i i peptides into active Identifies active process very large conformations compounds screens Informatics driven Generates diverse Extensive coverage of Identify active bioisosteres chemical space pharmacophore Enrichment cycle
- 22. BLOSUM62 matrix vs Field Based Substitution Matrix BLOSUM62 matrix vs Field Based Substitution Matrix FBSM10 BLOSUM62 New matrices more appropriate to PPIs
- 24. Cresset s Backbone vs Field Comparison Cresset’s Backbone vs Field Comparison 2D structural diversity of the HIV NNRTIs contrasts with strong Field similarity when structures are overlaid
- 25. Applications of CIS Display Protease cleavage site determination P t l it d t i ti Peptide stability Cell penetrating peptide discovery Scaffold engineering Scaffold engineering
- 26. CIS Display: Protease Site CIS Display: Protease Site Determination FLAG epitope random librar library ┌─────────────┐ D-Y-K-F-D-D-Y-W-H-x-x-x-x-x-x-x-x-x-x-LINKER-REPA Protease sensitive library peptides cleaved Genes encoding protease sensitive peptides lost ii id l
- 27. Protease Cleavage Specificity 1.2 +thrombin -thrombin 1 0.8 nm Abs450n 0.6 0.4 0.2 0 1 2 3 4 5 6 7 8 9 10 Control Novagen LVPR|GS 1H SWCHARLTPR|GS | CIS display‐ CIS di l 5E TVRPR|SNSNT identified 12C RGLLYLPR|RN thrombin 1-2B LCATTLGPR|S substrate s bstrate 1-3G GVPPRPR|ALS sequences 2-4H PR|AVSYLDVG
- 28. GPCR Ligand Stabilisation Stability maturation of native GPCR ligand Stability maturation of native GPCR ligand • Aim of project: select a peptide with increased stability in human plasma whilst maintaining activity and specificity over a membrane of related target. • Library based upon a consensus sequence derived from the wild type. • The target was presented as an overexpressing cell membrane fraction. Activity Stability in human plasma 80 100 WT peptide 90 Peptide X Peptide X Peptide X 60 80 Peptide Y roduction 70 activity 60 40 50 IP pr % a 40 30 20 20 WT peptide 10 0 0 0 ‐10 ‐9 ‐8 ‐7 ‐6 0 50 100 150 200 Peptide (log M) Time/min
- 29. Selection for Cell Penetrating Peptides DNA library encoding peptide RepA fusions Initial library Peptide‐RepA‐DNA complexes formed Lysis to release Isolate CPPs internalised complexes p DNase Digest/wash away exposed p protein‐DNA complexes p Protease
- 30. CPP Confocal Study Live CHO Cells CPP Confocal Study Live CHO Cells E5 Tat Control C l Peptide (green) + Annexin V (red) Transmitted light li h Peptide E5 labelled with N‐terminal (5/6)‐fluorescein
- 31. CIS Display and Proteins CIS display is suitable for the expression of larger protein fusions i fl t i f i Has been used for next generation p protein scaffolds and antibody y fragments Lysate has been developed to handle multiple cysteine containing h dl lti l t i t i i systems Licenced to Centocor for Centyrin y scaffold
- 32. Alternative scaffolds Betatein (Isogenica) B t t i (I i ) Can be recombinantly expressed or chemically synthesised y p y y Randomly mutate surface residues Less than 40 amino acids Less than 40 amino acids Reversible folding folded unfolded f ld d
- 33. Alternative scaffolds Betatein B t t i Selection from naïve library: affinity for VEGFR2 y y Best clone : KD = 24.0±4.0nM (ForteBio) Binding specificy 3.5 35 3.0 VEGFR2 huIgG mAb enzyme X sorbance 2.5 albumin neg 2.0 1.5 abs 1.0 0.5 0.0 A1 B1 E1 H4 H6 neg clone l
- 34. Isogenica Summary Isogenica Summary A cost effective biologics discovery platform combining rapid A cost effective biologics discovery platform combining rapid library display and next generation sequencing for multiple library formats y Amenable to high throughput automation Resulting data rich output enables design of maturation Resulting data rich output enables design of maturation libraries Rapid progression through maturation to lead identification Rapid progression through maturation to lead identification Display of peptides, protein scaffolds and antibody fragments and other proteins upto fragments and other proteins upto 90kDa Applicable to small molecule drug discovery through BIC collaboration
- 35. Partners
- 36. Thank Th k you Any Questions? www.isogenica.com Tel: +44 (0)1799 533680