High Throughput Discovery of Novel Peptides with Biological Function

F. Billwiller, G. Poloni, B. Marshall, X. Pan, X. Ni and A. Pitt

Orbit Discovery Ltd., Oxford, United Kingdom

© Orbit Discovery Ltd. 2019

Introduction ORBIT Peptide Display Platform

Summary

1. One piece of DNA from a randomised or focused library binds to each bead.

2. DNA amplification on beads within emulsion droplet.

3. Generation of peptides on beads within emulsion droplet. Genotype is linked to phenotype.

4. Screening library is incubated with target protein or cells.

5. DNA sequencing informs of the amino acid sequence of hits. Bioinformatics reveals recurring motifs.

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ORBIT Peptide Display - Targeting HIV

*Huang, L.-C., et al. (2013). Linking genotype to phenotype on beads: high throughput selection of peptides with biological function. Scientific Reports, 3, 1–9.

The ORBIT peptide display platform possesses the positiveattributes of traditional methods with the ability toaddress cell surface targets for the discovery of functionalpeptide therapeutic leads.

Comparison With Other Methods

Highly ranked peptides obtained by ORBIT bead-library screening on HIV-1 viral coat protein GP120were synthesised and tested by ELISA for GP120 binding. Four peptides with the highest affinity toGP120 were tested in a HIV-1 replication assay at three concentrations with or without streptavidinconjugation. Three HIV-1 strains (BAL, IIIB, clade C) were tested (positive control: T20, Enfuvirtide).

Surface plasmon resonance (Biacore) was used to measure the KD ofthe candidate peptides and GP120. The peptides were determined tohave KD values between 39 and 80 μM, the Kd calculation for GP16peptide is shown as an example.

• Orbit Discovery is a peptide display technology company foundedin 2016 as an Oxford University spin-out.

• Peptides are highly active biological molecules with great drugpotential, high specificity, low toxicity and low manufacturingcosts which currently constitute 2-5% of the drug market.

• The ORBIT peptide display platform is highly adaptable allowingfor enhancements by the adoption of cutting-edge technologies.

• The ORBIT Peptide Display Platform has all the positive attributes of phage display combined with the added flexibility of in vitro display methods.

• ORBIT beads can display cyclic peptides and incorporate unnatural amino acids.

• The platform is highly adaptable allowing for the adoption of cutting-edge technologies such as next generation DNA sequencing, microfluidics and FACS.

• ORBIT peptide display can be utilised for the in situ screening of cell surface targets to identify not just peptides that specifically bind to the target but also thosepeptides that bind and elicit a functional response from the target.

Whole Cell ScreeningCells expressing a target linked to a reporter systemallow for functional peptide hit identification in theprimary screen.

1. Individual cells are encapsulated in dropletsutilizing microfluidics technologies.

2. Fluorescent cells indicate GPCR activation bypeptides displayed on ORBIT beads.

1. Target + negative control 2. Target + Orbit beads

Via FACS we are able to identify and sort specificbinding peptides to one or multiple in solution targets.

1. Soluble target with negative control (scaffold beads)

2. Soluble target with ORBIT beads. Positive bindersshowing increased fluorescence are indicated ingreen.

Incorporation of Unnatural Amino Acids

Codon reassignment was used to incorporatefluorescent amino acids into ORBIT displayed peptides.

1. Synthetic tRNAs loaded with BODIPY-labelledcysteine and azido phenylalanine (AzF) were addedto an in vitro transcription/translation reactiondepleted of endogenous tRNAs. TAMRA-DIBO wasused to label AzF via click chemistry.

2. SDS-PAGE images of target proteins containingeither one (left lane) or the two (right lane)fluorescent amino acids are shown.

In Solution Screening

A: Merged imageB: 488nm channel for green fluorescence labelled non-natural amino acid 1C: 546nm channel for blue fluorescence labelled non-natural amino acid 2

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