Technology

The Bicycle technology is based on the ability to apply Darwinian selection to repertoires of chemically constrained peptides. The technology requires the creation of repertoires of peptides displayed on the surface of bacteriophage which can be modified with an organochemical scaffold to create a diverse array of constrained peptides. These repertoires can then be subjected to iterative selections to identify high affinity binding peptides.

Libraries of peptides consisting of two variable regions flanked by cysteine residues are created as fusion proteins with the P3 phage coat protein. This allows the creation of peptides with two binding loops when the fusion protein is displayed on the surface on the bacteriophage and subsequently modified with the organochemical scaffold.

Generation of peptide libraries

Chemical modification is performed by reaction of the bacteriophage with the halomethylarene, tris( bromomethyl) benzene(TBMB), to create a diverse library of highly constrained peptides. The modified phages are subjected to selection on antigen coated surfaces in order to enrich for antigen binding peptides. Multiple cycles of selection and modification are repeated to select for high affinity binding.

Selection of antigen binding constrained peptides

The serum protease kallikrein has been used as a target antigen. Using this antigen after several rounds of enrichment and selection, peptides were identified which inhibited the enzyme activity of of kallikrein with IC50’s between 20 and 100nM when synthesised as discrete peptides. Inhibitory activity was strictly dependent on the presence of the TBMB core as the linear peptides had much lower inhibitory activity compared to the constrained peptide. Sequencing of these peptides generated after these initial rounds of selection revealed that consensus peptide sequences had emerged in each of the two loops of the selected constrained peptides.

Sequences of phage displayed peptides after initial rounds of selection for kallikrein binding

Affinity maturation of the kallikrein specific peptides was achieved by the creation of new libraries in which the amino acids in one of the variable regions was randomised whilst retaining the consensus sequence in the other variable region. Further selections with these libraries allowed the selection of even higher potency peptides. For example, peptide PK15 has an IC50 of 1.7nM as a synthetic constrained peptide – an approximate 20 fold increase in potency.

Sequences after affinity maturation of clones

Structural modelling of the peptide complexes suggests that the loops in the bicyclic peptide are indeed constrained by the organochemical scaffold. This has similarities to the way in which the complementarity determining regions (CDRs ) are oriented by the non-variable regions of the antibody molecule.

The ability to apply Bicycle technology to a broad range of targets is being tested in order to understand the potential of this approach to create a new generation of therapeutic molecules which combine the advantages of biologics and NCEs.

If you would like to learn more about the technology please read the original scientific publication in Nature Chemical Biology vol. 5 pp 502-507 (2009).