Abstract 1352: Computational design of a cyclic peptide that inhibits the CTLA4 in T cells and the growth of lung carcinoma in mice

Abstract

Abstract Proteins involved in immune checkpoint pathways, such as CTLA4, PD-1, and PD-L1, have become important targets for cancer immunotherapy; however, the 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 designed 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 lung cancer mouse model in immunocompetent mice. Beginning from a template excised from the x-ray structure of the CTLA4:B7-2 complex, we generate several peptide sequences using Rosetta, a protein modelling program. 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 Binding Free Energy Estimator (BFEE). The most promising peptide, cyc-EIDTVLTPTGWVAKRYS (hereafter CTLA4-ip), yields the standard free energy -6.6 ± 3.5 kcal/mol, which corresponds to a dissociation constant of 15 μmol/L. The binding affinity of this peptide for CTLA4 is measured experimentally (31 ± 4 μmol/L) using bio-layer interferometry. Pharmacokinetics of CTLA4-ip in a cell culture with Lewis lung carcinoma (LLC) cells and in mice revealed that this peptide is significantly more stable in both cell culture and in mice as compared to a control non-cyclic scrambled-sequence. Treatment with CTLA4-ip inhibited cancer cell growth in a co-culture of LLC cells and LLC cell-antigen primed murine T cells. Intraperitoneal administration of the CTLA4-ip (10mg/kg/day, every other day totaling 4 doses) markedly inhibited lung tumor growth in mice with an orthotropic LLC allograft model. Efficacy of the tumor growth inhibition by the CTLA4-ip was similar to that by an anti-PD-L1 antibody (10mg/kg/day, every two days totaling 3 doses). These results strongly suggest that this novel CTLA4-ip works as an immune checkpoint inhibitor to CTLA4 and is usable for lung cancer treatment. This research was supported by Kansas State University Johnson Cancer Research center (MT), Midwest Biomedical Accelerator Consortium (MT, JC), National Cancer Institute MT, JC) and the National Science Foundation (JC). Citation Format: Ravindra Thakkar, Deepa Upreti, Susumu Ishiguro, Geraldine Magnin, Keshari Sudasinghe, Anabelle Hall, Sarah DeVader, Masaaki Tamura, Jeffrey Comer. Computational design of a cyclic peptide that inhibits the CTLA4 in T cells and the growth of lung carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1352.