GFP-like protein scaffolds for antigen grafting: Soluble and stable neoantigen display in mammalian-compatible platforms

Abstract

Subunit vaccines are often limited by poor solubility and structural instability, affecting their ability to induce strong immune responses. Scaffold-based antigen display offers a promising solution, but translating designs into stable, scalable proteins remains challenging. Here, we present a pipeline for selecting and inserting tumor-derived peptide sequences into ß-barrel protein frameworks, using both GFP and the structurally similar G2 domain of the mouse nidogen. We validate the ASP173 loop in GFP as an effective insertion site, accommodating a double-peptide construct, as well as two distinct single peptides, without compromising folding or fluorescence. We further show that this design logic extends to the mouse nidogen G2 domain, which we employ as a host-native scaffold to carry the inserted epitope. The resulting constructs retain structural integrity and thermal stability, supporting their use in preclinical models and facilitating future applications in human health. This work establishes a modular, mammalian-compatible platform for stable tumor-associated peptide presentation, thereby advancing the development of next-generation cancer vaccines.

The study was mainly funded by the Agencia Española de Investigación (AEI) through the project PID2022-136845OB-10/AEI/10.13039/501100011033 to E.V. and by AGAUR through the project SGR 2021 00092 to A.V. We would like to thank the ISCIII for funding CIBER-BBN (CB06/01/0014 and NANOREMOTE) and the Ministry of Science for funding the ICTS Platform Nanbiosis. The production of the fusion proteins was assisted, in part, by the Protein Production (PPP) Unit of the Platform Nanbiosis of the CIBER-BBN/IBB. Molecular graphics were performed with UCSF Chimera and ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from the National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. ESI-TOF and ICP-MS experiments were performed at Servei d'Anàlisi Química (SAQ) in Universitat Autònoma de Barcelona (UAB). Fluorimetry experiments were performed at Servei de Genòmica i Espectroscòpia de Biomolècules (SGiEB) in UAB. Electron microscopy was performed at the Servei de Microscòpia i Difracció de Raigs X (UAB). MALDI-TOF experiments were performed at Unitat d'Espectrometria de Masses de Caracterització Molecular CCiTUB in Universitat de Barcelona (UB).

Peer Reviewed

Postprint (published version)