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

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 β-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.