Isogenica Ltd Biologics discovery overview 1
Isogenica Specialist antibody and peptide display company Based in Cambridge at the heart of UK Biotech Expertise in protein and peptide engineering since 2001 Offering a range of leading edge library and display technologies Flexible partnering models 2
Key components for antibody libraries Library design Avoid liabilities Incorporate advantages 100 90 80 70 60 50 40 30 20 10 0 Library generation Library synthesis with Colibra Design-to-functional library 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Y W V T S R Q P N M L K I H G F E D C A Library display Proprietary CIS Display (cell-free) Eukaryotic display (H2 2015) Phage display 3
Library diversity from natural repertoire 10 14 Theoretical IgG naïve V(D)J repertoire 10 13 10 12 10 11 Total B-cells in an adult 10 10 10 9 10 8 10 7 Non-redundant PBMC naïve clones/adult 10 6 10 5 Non-redundant PBMC IgG clones/adult 4
Diversity vs functionality Natural repertoire libraries give high functionality but low diversity Poor quality synthetic libraries give high diversity but low functionality because of design issues, Random synthetic diversity is not tolerated in many positions in the CDRs Further aggravated as the length of the CDRs increase and build issues Conventional degenerate oligos lead to coding defects (stop codons, frameshifts, etc) Tri-nucleotide phosphoramidate (TRIM) oligos lack fine control of amino acid representation 5
Analysis of the natural repertoire Through analysis of the natural repertoire we now: Understand the natural positional bias in amino acid use Understand how CDR length influences this bias Understand which are the optimal frameworks for use This results in the capacity to design synthetic libraries with high functional diversity 6
Translating the theoretical library into the library design Optimal functional diversity Optimal framework choice Converts theoretical library into practical library design Optimal biochemical characteristics Optimal dispersion of diversity 7
Colibra technology Blunt ended ligation methodology Codons selected for optimal performance Integration with next generation sequencing for full functional and sequence based quality control Automation for scale-up CODON LIBRARY ACCEPTOR FRAMEWORK 5 3 ----- ----- 3 5 5 P 3 3 Ligate and Amplify 5 5 3 ----- ----- Digest with Type IIS RE 3 5 5 3 Repeat, next cycle 3 5 8
Advantages of Colibra Enables precise library build, humanisation, affinity maturation, natural repertoire mimicry Colibra TM allows codon pairs to be defined and accurately incorporated Reduce liabilities (isomerisation, deamidation, glycosylation, protease sites, oxidation sites, cysteine) Colibra TM libraries are true to the original design Improves efficiency of antibody discovery projects 9
CIS display cell-free DNA display RepA is a naturally occurring protein that binds its own DNA sequence allowing it to act as a linker between phenotype and genotype 10
Advantages of CIS display Bind to target No transformation, unlike phage display DNA-based system, not RNA Cis-activity of DNA-binding protein RepA Libraries up to 10 13 expressed in vitro stable protein-dna complexes Protein-DNA complexes subjected to affinity selection Eluted complexes regenerated by PCR Optimal ligands after 3-5 successive rounds Robust methodology and easy technology transfer 11
V HH antibody fragment Single domain of camelid heavy chain only antibody Maintains specificity and affinity of parent antibody Validated as therapeutic in clinical trials Small (12-15 kda) High ph and temperature stability Simple, cost-effective manufacturing Ideal for ADC and multivalent formatting heavy chain IgG 12
Conventional route to generating V HH s heavy chain IgG Immunise a llama Extract blood Clone PCR Minimum 10 weeks work Labour intensive Accessed 10 6-10 7 diversity 13
Using the llamda TM system in CIS display Integrating: Powerful design Precision built through Colibra CIS display automatable in vitro system Library size > 10 12 unique domains (a million llamas in a tube) Hits generated in 4-6 weeks Nanomolar affinities from naïve selections 14
Synthetic human scfv libraries: Xab TM system Built through Colibra TM IgG conversion scfv Operated in phage display Available for service and licensing VH VL VH CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 natural synthetic natural natural synthetic synthetic natural natural synthetic natural synthetic synthetic VL Libraries Athos Porthos Aramis Tested in phage 3 sub-libraries 3 sub-libraries 2 sub-libraries synthetic synthetic 15
4D Fab TM library construction - 2015 IgG conversion Fab Design by world class expertise in computational immunology Best-in-class functionality expected to be >Ylanthia (Morphosys) Fabrication initiated Early partnerships sought 16
Partnership in biologics discovery NextGen synthetic antibody libraries Discovery technologies VHH, scfv, Fab Maximise functional diversity Range of display technologies Scaffold and peptide libraries available Partnering CIS display Phage display IgG conversion Next generation sequencing Bioinformatics Analytical screening Discovery partnerships Lead optimisation partnerships Access to proprietary libraries Access to display technologies Cell based screens Flow cytometry / Cell sorting Affinity determination Biophysical analyses 17
Isogenica Ltd Biologics discovery overview 18