Computational design of a cyclic peptide that inhibits the CTLA4 immune checkpoint.

Proteins involved in immune checkpoint pathways, such as CTLA4, PD1, and PD-L1, have become important targets for cancer immunotherapy; however, development of small molecule drugs targeting these pathways has proven difficult due to the nature of their protein-protein interfaces. Here, using a hierarchy of computational techniques, we design a cyclic peptide that binds CTLA4 and follow this with experimental verification of binding and biological activity, using bio-layer interferometry, cell culture, and a mouse tumor model. Beginning from a template excised from the X-ray structure of the CTLA4:B7-2 complex, we generate several peptide sequences using flexible docking and modeling steps. These peptides are cyclized head-to-tail to improve structural and proteolytic stability and screened using molecular dynamics simulation and MM-GBSA calculation. The standard binding free energies for shortlisted peptides are then calculated in explicit-solvent simulation using a rigorous multistep technique. The most promising peptide, cyc(EIDTVLTPTGWVAKRYS), yields the standard free energy -6.6 ± 3.5 kcal mol -1 , which corresponds to a dissociation constant of ∼15 μmol L -1 . The binding affinity of this peptide for CTLA4 is measured experimentally (31 ± 4 μmol L -1 ) using bio-layer interferometry. Treatment with this peptide inhibited tumor growth in a co-culture of Lewis lung carcinoma (LLC) cells and antigen primed T cells, as well as in mice with an orthotropic Lewis lung carcinoma allograft model.