Protein Engineering for Personalized therapy and diagnosis

Protein Engineering, especially protein post-translational modification (PTM), ex-tends proteomes in a more complex way than one can expect from analysis of their encod-ing genomes. It can activate or deactivate certain catalytic functions, add new desired func-tions, or change some biological activity of the protein. In this thesis work, we use protein engineering tools to show that a number of functions can be engineered to improve protein-based therapeutics. First, on melanoma cancer vaccine development, we demonstrated a new and ver-satile nanovaccine platform to address the major challenges in neoantigen cancer vaccine delivery by "polymerizing" the neoepitopes through a reversible polycondensation reaction. Using synthetic long peptide (SLP) bearing a neoepitope and multiple amine groups as one monomer (monomer A) mixed with another reactive bi-functional monomer (monomer B), we prepared a polycondensate neoepitope (PNE) with controlled sizes and responsiveness, which showed superior LN targeting and efficient activation of antigen-presenting cells (APCs). Upon internalization by APCs, redox-responsiveness antigen release facilitated the endosome escape and cytosol delivery of peptide antigens and markedly promoted the cross-presentation, and elicited potent antigen-specific CD8+ T cell responses in immunized mice, therefore, enabling markedly enhanced antitumor efficacy in a prophylactic mouse model. Second, on antivirals development, we demonstrated a protein-based new and ver-satile approach for broad-spectrum virucidal material through a one-step reaction by simply chemically conjugating a long flexible and hydrophobic ligand onto the surface of a protein core. Modified proteins reproducibly showed not only effective antiviral inhibition but also a good virucidal effect. Broad-spectrum antiviral inhibition effect was observed against HSV-2, Influenza H1N1, and SARS-CoV-2. Two important key factors, ligand density, and ligand hydrophobic force, significantly influenced antiviral inhibition and virucidal effect. This protein-based antiviral platform provided an easy-manufactured, versatile, broad-spectrum effective, and potentially translatable antiviral solution. At last, we demonstrated a lipoprotein-cholesterol nanoparticle-based non-invasive cancer diagnosis system. In this preliminary test and proof of concept, lipoprotein-cholesterol nanoparticles were extracted from 5 melanoma cancer patients' serum by a lab-developed simple and reproducible technique with a high yield. This method successfully eliminated the most abundant inert protein serum albumin and accumulated low abundance proteins, which are usually masked by serum albumins. LC-MS/MS proteomic analysis data indicates within a false discovery rate less than 0.05, differentially expressed proteins, either up-or down-regulated proteins with fold-change over 2, were identified and can potentially be used as cancer biomarkers.